Source Name	Comment [Sample_description]	Comment [Sample_description]	Comment [Sample_description]	Comment [Sample_description]	Comment [Sample_description]	Comment [Sample_description]	Comment [Sample_description]	Comment [Sample_description]	Comment [Sample_description]	Comment [Sample_source_name]	Characteristics[Organism]	Extract Name	Material Type	Labeled Extract Name	Label	Assay Name	Technology Type	Comment [Platform_title]	Protocol REF	Normalization Name	Derived Array Data File	Comment [Derived ArrayExpress FTP file]
GSM38888 1	Large-scale temporal gene expression profiling during gonadal differentiation and early gametogenesis in rainbow-trout	Animals and samplings:	Research involving animal experimentation has been approved by the author's institution (authorization number 35-14) and conforms to NIH guidelines. All-male and all-female rainbow trout populations were obtained from the INRA experimental fish farm (Drennec, France) using breeders from the Mirwart strain. At 55 days post-fertilization (55 dpf, i.e., the onset of the free swimming), a male and female batch of 1500 fry each were transferred in 0.3 m3 tanks with recirculating water conditions. They were held at 12°C under constant photoperiod (12h light : 12h dark) and fed ad libitum with a commercial diet (dry pellet food, BiomarTM, Brande, Denmark). In each group, from 20 to 100 gonads, depending on the age of the fish, were sampled and pooled in duplicates at various stages of development : the onset of the free swimming period after complete yolk resumption (Day 0 = D0), D7, D12 (around the first occurrence of oocyte meiosis), D27 (first ovarian lamellar structures), D60 (first previtellogenic oocytes), D90 and D110. There were immediately frozen in liquid nitrogen and stored at -80°C until RNA extraction. Additional gonads were sampled at the same dates for histological analysis performed as previously described.	Total RNA extraction and Reverse Transcription:	Total RNA was extracted using TRIzol reagent (Invitrogen, Cergy Pontoise, France). Total RNA concentration was determined with an Agilent 2100 Bioanalyzer and the RNA 6000 LabChip kit (Agilent Technologies, Stockport, UK) according to the manufacturer's instructions. For cDNA synthesis, 1µg of RNA was denaturated in the presence of random hexamers (0.5µg) for 5 min at 70°C, and then chilled on ice. Reverse transcription (RT) was performed at 37°C for 1 h using M-MLV reverse transcriptase (Promega, Madison, WI) as described by the manufacturer.	Primers design:	Trout candidate gene homologues were searched in international public databanks using a reciprocal blast hit strategy. Candidate genes were chosen according to a bibliographic analysis showing their direct or indirect involvement in the sex differentiation cascade in vertebrates. All the primers were purchased from Eurogentec (Eurogentec, Seraing, Belgium). These primers were manually designed respecting whenever possible the following restriction parameters : 21-23 bp length, no more than 4 identical successive nucleotides, 30-70% GC content, a maximum of 2 G or C among the five 3'-end bases, no primer dimers and a short amplicon size (70-150 bp). Secondary structures were searched with DNA mfold (http://bioinfo.math.rpi.edu/~mfold/DNA/form1.cgi). Whenever possible, each pair was chosen with at least one primer flanking an intron-exon boundary, in order to prevent genomic amplification.	Real-time RT-PCR	Real-time RT-PCR was carried out on an iCycler iQTM (BioRad, Hercules, CA). Reactions were performed in 20µl with 300nM of each primer, 5µl of a 1/50 dilution of the RT reaction and the SYBER-Green PCR master Mix (Eurogentec) according to the manufacturer's instructions. After two incubation steps (50°C 10 min, 95°C 2 min), the thermal cycling protocol was 95°C for 10 min followed by 40 cycles of PCR (95°C 30 s, 60°C or 65°C 1 min). For each primer set the efficiency of the PCR reaction (linear equation : y = slope + intercept) was measured in triplicate on serial dilutions of the same cDNA sample (pool of reverse transcribed RNA samples). Real-time PCR efficiencies for each reaction were then calculated using the formula : Efficiency (E)=[10(1/slope)]-1. Melting curve analysis was also performed for each gene to check the specificity and identity of the RT-PCR products. The relative amount of the target RNA called the Starting Quantity (SQ) was then determined using the I-Cycler IQ software by comparison with the corresponding standard curve for each sample run in duplicate. SQ were calculated as follow : SQ = [10((Ct -intercept)/slope))]-1 where Ct is the Cycle threshold of the unknown sample. Each transcript level was then normalized by division with the expression values of the constitutive elongation factor 1 alpha (EF1a) used as an internal standard.	Male J27 - 1	Oncorhynchus mykiss	GSM38888 extract 1	total RNA	GSM38888 LE 1	1	GSM38888	assay	Rainbow trout sex differentiation cascade-related genes investigated using RT-PCR	P-GSE2151-1	GSM38888_sample_table.txt norm	GSM38888_sample_table.txt	ftp://ftp.ebi.ac.uk/pub/databases/microarray/data/experiment/GEOD/E-GEOD-2151/E-GEOD-2151.processed.1.zip
GSM38887 1	Large-scale temporal gene expression profiling during gonadal differentiation and early gametogenesis in rainbow-trout	Animals and samplings:	Research involving animal experimentation has been approved by the author's institution (authorization number 35-14) and conforms to NIH guidelines. All-male and all-female rainbow trout populations were obtained from the INRA experimental fish farm (Drennec, France) using breeders from the Mirwart strain. At 55 days post-fertilization (55 dpf, i.e., the onset of the free swimming), a male and female batch of 1500 fry each were transferred in 0.3 m3 tanks with recirculating water conditions. They were held at 12°C under constant photoperiod (12h light : 12h dark) and fed ad libitum with a commercial diet (dry pellet food, BiomarTM, Brande, Denmark). In each group, from 20 to 100 gonads, depending on the age of the fish, were sampled and pooled in duplicates at various stages of development : the onset of the free swimming period after complete yolk resumption (Day 0 = D0), D7, D12 (around the first occurrence of oocyte meiosis), D27 (first ovarian lamellar structures), D60 (first previtellogenic oocytes), D90 and D110. There were immediately frozen in liquid nitrogen and stored at -80°C until RNA extraction. Additional gonads were sampled at the same dates for histological analysis performed as previously described.	Total RNA extraction and Reverse Transcription:	Total RNA was extracted using TRIzol reagent (Invitrogen, Cergy Pontoise, France). Total RNA concentration was determined with an Agilent 2100 Bioanalyzer and the RNA 6000 LabChip kit (Agilent Technologies, Stockport, UK) according to the manufacturer's instructions. For cDNA synthesis, 1µg of RNA was denaturated in the presence of random hexamers (0.5µg) for 5 min at 70°C, and then chilled on ice. Reverse transcription (RT) was performed at 37°C for 1 h using M-MLV reverse transcriptase (Promega, Madison, WI) as described by the manufacturer.	Primers design:	Trout candidate gene homologues were searched in international public databanks using a reciprocal blast hit strategy. Candidate genes were chosen according to a bibliographic analysis showing their direct or indirect involvement in the sex differentiation cascade in vertebrates. All the primers were purchased from Eurogentec (Eurogentec, Seraing, Belgium). These primers were manually designed respecting whenever possible the following restriction parameters : 21-23 bp length, no more than 4 identical successive nucleotides, 30-70% GC content, a maximum of 2 G or C among the five 3'-end bases, no primer dimers and a short amplicon size (70-150 bp). Secondary structures were searched with DNA mfold (http://bioinfo.math.rpi.edu/~mfold/DNA/form1.cgi). Whenever possible, each pair was chosen with at least one primer flanking an intron-exon boundary, in order to prevent genomic amplification.	Real-time RT-PCR	Real-time RT-PCR was carried out on an iCycler iQTM (BioRad, Hercules, CA). Reactions were performed in 20µl with 300nM of each primer, 5µl of a 1/50 dilution of the RT reaction and the SYBER-Green PCR master Mix (Eurogentec) according to the manufacturer's instructions. After two incubation steps (50°C 10 min, 95°C 2 min), the thermal cycling protocol was 95°C for 10 min followed by 40 cycles of PCR (95°C 30 s, 60°C or 65°C 1 min). For each primer set the efficiency of the PCR reaction (linear equation : y = slope + intercept) was measured in triplicate on serial dilutions of the same cDNA sample (pool of reverse transcribed RNA samples). Real-time PCR efficiencies for each reaction were then calculated using the formula : Efficiency (E)=[10(1/slope)]-1. Melting curve analysis was also performed for each gene to check the specificity and identity of the RT-PCR products. The relative amount of the target RNA called the Starting Quantity (SQ) was then determined using the I-Cycler IQ software by comparison with the corresponding standard curve for each sample run in duplicate. SQ were calculated as follow : SQ = [10((Ct -intercept)/slope))]-1 where Ct is the Cycle threshold of the unknown sample. Each transcript level was then normalized by division with the expression values of the constitutive elongation factor 1 alpha (EF1a) used as an internal standard.	Male J12 - 2	Oncorhynchus mykiss	GSM38887 extract 1	total RNA	GSM38887 LE 1	1	GSM38887	assay	Rainbow trout sex differentiation cascade-related genes investigated using RT-PCR	P-GSE2151-1	GSM38887_sample_table.txt norm	GSM38887_sample_table.txt	ftp://ftp.ebi.ac.uk/pub/databases/microarray/data/experiment/GEOD/E-GEOD-2151/E-GEOD-2151.processed.1.zip
GSM38886 1	Large-scale temporal gene expression profiling during gonadal differentiation and early gametogenesis in rainbow-trout	Animals and samplings:	Research involving animal experimentation has been approved by the author's institution (authorization number 35-14) and conforms to NIH guidelines. All-male and all-female rainbow trout populations were obtained from the INRA experimental fish farm (Drennec, France) using breeders from the Mirwart strain. At 55 days post-fertilization (55 dpf, i.e., the onset of the free swimming), a male and female batch of 1500 fry each were transferred in 0.3 m3 tanks with recirculating water conditions. They were held at 12°C under constant photoperiod (12h light : 12h dark) and fed ad libitum with a commercial diet (dry pellet food, BiomarTM, Brande, Denmark). In each group, from 20 to 100 gonads, depending on the age of the fish, were sampled and pooled in duplicates at various stages of development : the onset of the free swimming period after complete yolk resumption (Day 0 = D0), D7, D12 (around the first occurrence of oocyte meiosis), D27 (first ovarian lamellar structures), D60 (first previtellogenic oocytes), D90 and D110. There were immediately frozen in liquid nitrogen and stored at -80°C until RNA extraction. Additional gonads were sampled at the same dates for histological analysis performed as previously described.	Total RNA extraction and Reverse Transcription:	Total RNA was extracted using TRIzol reagent (Invitrogen, Cergy Pontoise, France). Total RNA concentration was determined with an Agilent 2100 Bioanalyzer and the RNA 6000 LabChip kit (Agilent Technologies, Stockport, UK) according to the manufacturer's instructions. For cDNA synthesis, 1µg of RNA was denaturated in the presence of random hexamers (0.5µg) for 5 min at 70°C, and then chilled on ice. Reverse transcription (RT) was performed at 37°C for 1 h using M-MLV reverse transcriptase (Promega, Madison, WI) as described by the manufacturer.	Primers design:	Trout candidate gene homologues were searched in international public databanks using a reciprocal blast hit strategy. Candidate genes were chosen according to a bibliographic analysis showing their direct or indirect involvement in the sex differentiation cascade in vertebrates. All the primers were purchased from Eurogentec (Eurogentec, Seraing, Belgium). These primers were manually designed respecting whenever possible the following restriction parameters : 21-23 bp length, no more than 4 identical successive nucleotides, 30-70% GC content, a maximum of 2 G or C among the five 3'-end bases, no primer dimers and a short amplicon size (70-150 bp). Secondary structures were searched with DNA mfold (http://bioinfo.math.rpi.edu/~mfold/DNA/form1.cgi). Whenever possible, each pair was chosen with at least one primer flanking an intron-exon boundary, in order to prevent genomic amplification.	Real-time RT-PCR	Real-time RT-PCR was carried out on an iCycler iQTM (BioRad, Hercules, CA). Reactions were performed in 20µl with 300nM of each primer, 5µl of a 1/50 dilution of the RT reaction and the SYBER-Green PCR master Mix (Eurogentec) according to the manufacturer's instructions. After two incubation steps (50°C 10 min, 95°C 2 min), the thermal cycling protocol was 95°C for 10 min followed by 40 cycles of PCR (95°C 30 s, 60°C or 65°C 1 min). For each primer set the efficiency of the PCR reaction (linear equation : y = slope + intercept) was measured in triplicate on serial dilutions of the same cDNA sample (pool of reverse transcribed RNA samples). Real-time PCR efficiencies for each reaction were then calculated using the formula : Efficiency (E)=[10(1/slope)]-1. Melting curve analysis was also performed for each gene to check the specificity and identity of the RT-PCR products. The relative amount of the target RNA called the Starting Quantity (SQ) was then determined using the I-Cycler IQ software by comparison with the corresponding standard curve for each sample run in duplicate. SQ were calculated as follow : SQ = [10((Ct -intercept)/slope))]-1 where Ct is the Cycle threshold of the unknown sample. Each transcript level was then normalized by division with the expression values of the constitutive elongation factor 1 alpha (EF1a) used as an internal standard.	Male J12 - 1	Oncorhynchus mykiss	GSM38886 extract 1	total RNA	GSM38886 LE 1	1	GSM38886	assay	Rainbow trout sex differentiation cascade-related genes investigated using RT-PCR	P-GSE2151-1	GSM38886_sample_table.txt norm	GSM38886_sample_table.txt	ftp://ftp.ebi.ac.uk/pub/databases/microarray/data/experiment/GEOD/E-GEOD-2151/E-GEOD-2151.processed.1.zip
GSM38885 1	Large-scale temporal gene expression profiling during gonadal differentiation and early gametogenesis in rainbow-trout	Animals and samplings:	Research involving animal experimentation has been approved by the author's institution (authorization number 35-14) and conforms to NIH guidelines. All-male and all-female rainbow trout populations were obtained from the INRA experimental fish farm (Drennec, France) using breeders from the Mirwart strain. At 55 days post-fertilization (55 dpf, i.e., the onset of the free swimming), a male and female batch of 1500 fry each were transferred in 0.3 m3 tanks with recirculating water conditions. They were held at 12°C under constant photoperiod (12h light : 12h dark) and fed ad libitum with a commercial diet (dry pellet food, BiomarTM, Brande, Denmark). In each group, from 20 to 100 gonads, depending on the age of the fish, were sampled and pooled in duplicates at various stages of development : the onset of the free swimming period after complete yolk resumption (Day 0 = D0), D7, D12 (around the first occurrence of oocyte meiosis), D27 (first ovarian lamellar structures), D60 (first previtellogenic oocytes), D90 and D110. There were immediately frozen in liquid nitrogen and stored at -80°C until RNA extraction. Additional gonads were sampled at the same dates for histological analysis performed as previously described.	Total RNA extraction and Reverse Transcription:	Total RNA was extracted using TRIzol reagent (Invitrogen, Cergy Pontoise, France). Total RNA concentration was determined with an Agilent 2100 Bioanalyzer and the RNA 6000 LabChip kit (Agilent Technologies, Stockport, UK) according to the manufacturer's instructions. For cDNA synthesis, 1µg of RNA was denaturated in the presence of random hexamers (0.5µg) for 5 min at 70°C, and then chilled on ice. Reverse transcription (RT) was performed at 37°C for 1 h using M-MLV reverse transcriptase (Promega, Madison, WI) as described by the manufacturer.	Primers design:	Trout candidate gene homologues were searched in international public databanks using a reciprocal blast hit strategy. Candidate genes were chosen according to a bibliographic analysis showing their direct or indirect involvement in the sex differentiation cascade in vertebrates. All the primers were purchased from Eurogentec (Eurogentec, Seraing, Belgium). These primers were manually designed respecting whenever possible the following restriction parameters : 21-23 bp length, no more than 4 identical successive nucleotides, 30-70% GC content, a maximum of 2 G or C among the five 3'-end bases, no primer dimers and a short amplicon size (70-150 bp). Secondary structures were searched with DNA mfold (http://bioinfo.math.rpi.edu/~mfold/DNA/form1.cgi). Whenever possible, each pair was chosen with at least one primer flanking an intron-exon boundary, in order to prevent genomic amplification.	Real-time RT-PCR	Real-time RT-PCR was carried out on an iCycler iQTM (BioRad, Hercules, CA). Reactions were performed in 20µl with 300nM of each primer, 5µl of a 1/50 dilution of the RT reaction and the SYBER-Green PCR master Mix (Eurogentec) according to the manufacturer's instructions. After two incubation steps (50°C 10 min, 95°C 2 min), the thermal cycling protocol was 95°C for 10 min followed by 40 cycles of PCR (95°C 30 s, 60°C or 65°C 1 min). For each primer set the efficiency of the PCR reaction (linear equation : y = slope + intercept) was measured in triplicate on serial dilutions of the same cDNA sample (pool of reverse transcribed RNA samples). Real-time PCR efficiencies for each reaction were then calculated using the formula : Efficiency (E)=[10(1/slope)]-1. Melting curve analysis was also performed for each gene to check the specificity and identity of the RT-PCR products. The relative amount of the target RNA called the Starting Quantity (SQ) was then determined using the I-Cycler IQ software by comparison with the corresponding standard curve for each sample run in duplicate. SQ were calculated as follow : SQ = [10((Ct -intercept)/slope))]-1 where Ct is the Cycle threshold of the unknown sample. Each transcript level was then normalized by division with the expression values of the constitutive elongation factor 1 alpha (EF1a) used as an internal standard.	Male J7 - 2	Oncorhynchus mykiss	GSM38885 extract 1	total RNA	GSM38885 LE 1	1	GSM38885	assay	Rainbow trout sex differentiation cascade-related genes investigated using RT-PCR	P-GSE2151-1	GSM38885_sample_table.txt norm	GSM38885_sample_table.txt	ftp://ftp.ebi.ac.uk/pub/databases/microarray/data/experiment/GEOD/E-GEOD-2151/E-GEOD-2151.processed.1.zip
GSM38884 1	Large-scale temporal gene expression profiling during gonadal differentiation and early gametogenesis in rainbow-trout	Animals and samplings:	Research involving animal experimentation has been approved by the author's institution (authorization number 35-14) and conforms to NIH guidelines. All-male and all-female rainbow trout populations were obtained from the INRA experimental fish farm (Drennec, France) using breeders from the Mirwart strain. At 55 days post-fertilization (55 dpf, i.e., the onset of the free swimming), a male and female batch of 1500 fry each were transferred in 0.3 m3 tanks with recirculating water conditions. They were held at 12°C under constant photoperiod (12h light : 12h dark) and fed ad libitum with a commercial diet (dry pellet food, BiomarTM, Brande, Denmark). In each group, from 20 to 100 gonads, depending on the age of the fish, were sampled and pooled in duplicates at various stages of development : the onset of the free swimming period after complete yolk resumption (Day 0 = D0), D7, D12 (around the first occurrence of oocyte meiosis), D27 (first ovarian lamellar structures), D60 (first previtellogenic oocytes), D90 and D110. There were immediately frozen in liquid nitrogen and stored at -80°C until RNA extraction. Additional gonads were sampled at the same dates for histological analysis performed as previously described.	Total RNA extraction and Reverse Transcription:	Total RNA was extracted using TRIzol reagent (Invitrogen, Cergy Pontoise, France). Total RNA concentration was determined with an Agilent 2100 Bioanalyzer and the RNA 6000 LabChip kit (Agilent Technologies, Stockport, UK) according to the manufacturer's instructions. For cDNA synthesis, 1µg of RNA was denaturated in the presence of random hexamers (0.5µg) for 5 min at 70°C, and then chilled on ice. Reverse transcription (RT) was performed at 37°C for 1 h using M-MLV reverse transcriptase (Promega, Madison, WI) as described by the manufacturer.	Primers design:	Trout candidate gene homologues were searched in international public databanks using a reciprocal blast hit strategy. Candidate genes were chosen according to a bibliographic analysis showing their direct or indirect involvement in the sex differentiation cascade in vertebrates. All the primers were purchased from Eurogentec (Eurogentec, Seraing, Belgium). These primers were manually designed respecting whenever possible the following restriction parameters : 21-23 bp length, no more than 4 identical successive nucleotides, 30-70% GC content, a maximum of 2 G or C among the five 3'-end bases, no primer dimers and a short amplicon size (70-150 bp). Secondary structures were searched with DNA mfold (http://bioinfo.math.rpi.edu/~mfold/DNA/form1.cgi). Whenever possible, each pair was chosen with at least one primer flanking an intron-exon boundary, in order to prevent genomic amplification.	Real-time RT-PCR	Real-time RT-PCR was carried out on an iCycler iQTM (BioRad, Hercules, CA). Reactions were performed in 20µl with 300nM of each primer, 5µl of a 1/50 dilution of the RT reaction and the SYBER-Green PCR master Mix (Eurogentec) according to the manufacturer's instructions. After two incubation steps (50°C 10 min, 95°C 2 min), the thermal cycling protocol was 95°C for 10 min followed by 40 cycles of PCR (95°C 30 s, 60°C or 65°C 1 min). For each primer set the efficiency of the PCR reaction (linear equation : y = slope + intercept) was measured in triplicate on serial dilutions of the same cDNA sample (pool of reverse transcribed RNA samples). Real-time PCR efficiencies for each reaction were then calculated using the formula : Efficiency (E)=[10(1/slope)]-1. Melting curve analysis was also performed for each gene to check the specificity and identity of the RT-PCR products. The relative amount of the target RNA called the Starting Quantity (SQ) was then determined using the I-Cycler IQ software by comparison with the corresponding standard curve for each sample run in duplicate. SQ were calculated as follow : SQ = [10((Ct -intercept)/slope))]-1 where Ct is the Cycle threshold of the unknown sample. Each transcript level was then normalized by division with the expression values of the constitutive elongation factor 1 alpha (EF1a) used as an internal standard.	Male J7 - 1	Oncorhynchus mykiss	GSM38884 extract 1	total RNA	GSM38884 LE 1	1	GSM38884	assay	Rainbow trout sex differentiation cascade-related genes investigated using RT-PCR	P-GSE2151-1	GSM38884_sample_table.txt norm	GSM38884_sample_table.txt	ftp://ftp.ebi.ac.uk/pub/databases/microarray/data/experiment/GEOD/E-GEOD-2151/E-GEOD-2151.processed.1.zip
GSM38883 1	Large-scale temporal gene expression profiling during gonadal differentiation and early gametogenesis in rainbow-trout	Animals and samplings:	Research involving animal experimentation has been approved by the author's institution (authorization number 35-14) and conforms to NIH guidelines. All-male and all-female rainbow trout populations were obtained from the INRA experimental fish farm (Drennec, France) using breeders from the Mirwart strain. At 55 days post-fertilization (55 dpf, i.e., the onset of the free swimming), a male and female batch of 1500 fry each were transferred in 0.3 m3 tanks with recirculating water conditions. They were held at 12°C under constant photoperiod (12h light : 12h dark) and fed ad libitum with a commercial diet (dry pellet food, BiomarTM, Brande, Denmark). In each group, from 20 to 100 gonads, depending on the age of the fish, were sampled and pooled in duplicates at various stages of development : the onset of the free swimming period after complete yolk resumption (Day 0 = D0), D7, D12 (around the first occurrence of oocyte meiosis), D27 (first ovarian lamellar structures), D60 (first previtellogenic oocytes), D90 and D110. There were immediately frozen in liquid nitrogen and stored at -80°C until RNA extraction. Additional gonads were sampled at the same dates for histological analysis performed as previously described.	Total RNA extraction and Reverse Transcription:	Total RNA was extracted using TRIzol reagent (Invitrogen, Cergy Pontoise, France). Total RNA concentration was determined with an Agilent 2100 Bioanalyzer and the RNA 6000 LabChip kit (Agilent Technologies, Stockport, UK) according to the manufacturer's instructions. For cDNA synthesis, 1µg of RNA was denaturated in the presence of random hexamers (0.5µg) for 5 min at 70°C, and then chilled on ice. Reverse transcription (RT) was performed at 37°C for 1 h using M-MLV reverse transcriptase (Promega, Madison, WI) as described by the manufacturer.	Primers design:	Trout candidate gene homologues were searched in international public databanks using a reciprocal blast hit strategy. Candidate genes were chosen according to a bibliographic analysis showing their direct or indirect involvement in the sex differentiation cascade in vertebrates. All the primers were purchased from Eurogentec (Eurogentec, Seraing, Belgium). These primers were manually designed respecting whenever possible the following restriction parameters : 21-23 bp length, no more than 4 identical successive nucleotides, 30-70% GC content, a maximum of 2 G or C among the five 3'-end bases, no primer dimers and a short amplicon size (70-150 bp). Secondary structures were searched with DNA mfold (http://bioinfo.math.rpi.edu/~mfold/DNA/form1.cgi). Whenever possible, each pair was chosen with at least one primer flanking an intron-exon boundary, in order to prevent genomic amplification.	Real-time RT-PCR	Real-time RT-PCR was carried out on an iCycler iQTM (BioRad, Hercules, CA). Reactions were performed in 20µl with 300nM of each primer, 5µl of a 1/50 dilution of the RT reaction and the SYBER-Green PCR master Mix (Eurogentec) according to the manufacturer's instructions. After two incubation steps (50°C 10 min, 95°C 2 min), the thermal cycling protocol was 95°C for 10 min followed by 40 cycles of PCR (95°C 30 s, 60°C or 65°C 1 min). For each primer set the efficiency of the PCR reaction (linear equation : y = slope + intercept) was measured in triplicate on serial dilutions of the same cDNA sample (pool of reverse transcribed RNA samples). Real-time PCR efficiencies for each reaction were then calculated using the formula : Efficiency (E)=[10(1/slope)]-1. Melting curve analysis was also performed for each gene to check the specificity and identity of the RT-PCR products. The relative amount of the target RNA called the Starting Quantity (SQ) was then determined using the I-Cycler IQ software by comparison with the corresponding standard curve for each sample run in duplicate. SQ were calculated as follow : SQ = [10((Ct -intercept)/slope))]-1 where Ct is the Cycle threshold of the unknown sample. Each transcript level was then normalized by division with the expression values of the constitutive elongation factor 1 alpha (EF1a) used as an internal standard.	Male J0 - 2	Oncorhynchus mykiss	GSM38883 extract 1	total RNA	GSM38883 LE 1	1	GSM38883	assay	Rainbow trout sex differentiation cascade-related genes investigated using RT-PCR	P-GSE2151-1	GSM38883_sample_table.txt norm	GSM38883_sample_table.txt	ftp://ftp.ebi.ac.uk/pub/databases/microarray/data/experiment/GEOD/E-GEOD-2151/E-GEOD-2151.processed.1.zip
GSM38882 1	Large-scale temporal gene expression profiling during gonadal differentiation and early gametogenesis in rainbow-trout	Animals and samplings:	Research involving animal experimentation has been approved by the author's institution (authorization number 35-14) and conforms to NIH guidelines. All-male and all-female rainbow trout populations were obtained from the INRA experimental fish farm (Drennec, France) using breeders from the Mirwart strain. At 55 days post-fertilization (55 dpf, i.e., the onset of the free swimming), a male and female batch of 1500 fry each were transferred in 0.3 m3 tanks with recirculating water conditions. They were held at 12°C under constant photoperiod (12h light : 12h dark) and fed ad libitum with a commercial diet (dry pellet food, BiomarTM, Brande, Denmark). In each group, from 20 to 100 gonads, depending on the age of the fish, were sampled and pooled in duplicates at various stages of development : the onset of the free swimming period after complete yolk resumption (Day 0 = D0), D7, D12 (around the first occurrence of oocyte meiosis), D27 (first ovarian lamellar structures), D60 (first previtellogenic oocytes), D90 and D110. There were immediately frozen in liquid nitrogen and stored at -80°C until RNA extraction. Additional gonads were sampled at the same dates for histological analysis performed as previously described.	Total RNA extraction and Reverse Transcription:	Total RNA was extracted using TRIzol reagent (Invitrogen, Cergy Pontoise, France). Total RNA concentration was determined with an Agilent 2100 Bioanalyzer and the RNA 6000 LabChip kit (Agilent Technologies, Stockport, UK) according to the manufacturer's instructions. For cDNA synthesis, 1µg of RNA was denaturated in the presence of random hexamers (0.5µg) for 5 min at 70°C, and then chilled on ice. Reverse transcription (RT) was performed at 37°C for 1 h using M-MLV reverse transcriptase (Promega, Madison, WI) as described by the manufacturer.	Primers design:	Trout candidate gene homologues were searched in international public databanks using a reciprocal blast hit strategy. Candidate genes were chosen according to a bibliographic analysis showing their direct or indirect involvement in the sex differentiation cascade in vertebrates. All the primers were purchased from Eurogentec (Eurogentec, Seraing, Belgium). These primers were manually designed respecting whenever possible the following restriction parameters : 21-23 bp length, no more than 4 identical successive nucleotides, 30-70% GC content, a maximum of 2 G or C among the five 3'-end bases, no primer dimers and a short amplicon size (70-150 bp). Secondary structures were searched with DNA mfold (http://bioinfo.math.rpi.edu/~mfold/DNA/form1.cgi). Whenever possible, each pair was chosen with at least one primer flanking an intron-exon boundary, in order to prevent genomic amplification.	Real-time RT-PCR	Real-time RT-PCR was carried out on an iCycler iQTM (BioRad, Hercules, CA). Reactions were performed in 20µl with 300nM of each primer, 5µl of a 1/50 dilution of the RT reaction and the SYBER-Green PCR master Mix (Eurogentec) according to the manufacturer's instructions. After two incubation steps (50°C 10 min, 95°C 2 min), the thermal cycling protocol was 95°C for 10 min followed by 40 cycles of PCR (95°C 30 s, 60°C or 65°C 1 min). For each primer set the efficiency of the PCR reaction (linear equation : y = slope + intercept) was measured in triplicate on serial dilutions of the same cDNA sample (pool of reverse transcribed RNA samples). Real-time PCR efficiencies for each reaction were then calculated using the formula : Efficiency (E)=[10(1/slope)]-1. Melting curve analysis was also performed for each gene to check the specificity and identity of the RT-PCR products. The relative amount of the target RNA called the Starting Quantity (SQ) was then determined using the I-Cycler IQ software by comparison with the corresponding standard curve for each sample run in duplicate. SQ were calculated as follow : SQ = [10((Ct -intercept)/slope))]-1 where Ct is the Cycle threshold of the unknown sample. Each transcript level was then normalized by division with the expression values of the constitutive elongation factor 1 alpha (EF1a) used as an internal standard.	Female J110 - 2	Oncorhynchus mykiss	GSM38882 extract 1	total RNA	GSM38882 LE 1	1	GSM38882	assay	Rainbow trout sex differentiation cascade-related genes investigated using RT-PCR	P-GSE2151-1	GSM38882_sample_table.txt norm	GSM38882_sample_table.txt	ftp://ftp.ebi.ac.uk/pub/databases/microarray/data/experiment/GEOD/E-GEOD-2151/E-GEOD-2151.processed.1.zip
GSM38881 1	Large-scale temporal gene expression profiling during gonadal differentiation and early gametogenesis in rainbow-trout	Animals and samplings:	Research involving animal experimentation has been approved by the author's institution (authorization number 35-14) and conforms to NIH guidelines. All-male and all-female rainbow trout populations were obtained from the INRA experimental fish farm (Drennec, France) using breeders from the Mirwart strain. At 55 days post-fertilization (55 dpf, i.e., the onset of the free swimming), a male and female batch of 1500 fry each were transferred in 0.3 m3 tanks with recirculating water conditions. They were held at 12°C under constant photoperiod (12h light : 12h dark) and fed ad libitum with a commercial diet (dry pellet food, BiomarTM, Brande, Denmark). In each group, from 20 to 100 gonads, depending on the age of the fish, were sampled and pooled in duplicates at various stages of development : the onset of the free swimming period after complete yolk resumption (Day 0 = D0), D7, D12 (around the first occurrence of oocyte meiosis), D27 (first ovarian lamellar structures), D60 (first previtellogenic oocytes), D90 and D110. There were immediately frozen in liquid nitrogen and stored at -80°C until RNA extraction. Additional gonads were sampled at the same dates for histological analysis performed as previously described.	Total RNA extraction and Reverse Transcription:	Total RNA was extracted using TRIzol reagent (Invitrogen, Cergy Pontoise, France). Total RNA concentration was determined with an Agilent 2100 Bioanalyzer and the RNA 6000 LabChip kit (Agilent Technologies, Stockport, UK) according to the manufacturer's instructions. For cDNA synthesis, 1µg of RNA was denaturated in the presence of random hexamers (0.5µg) for 5 min at 70°C, and then chilled on ice. Reverse transcription (RT) was performed at 37°C for 1 h using M-MLV reverse transcriptase (Promega, Madison, WI) as described by the manufacturer.	Primers design:	Trout candidate gene homologues were searched in international public databanks using a reciprocal blast hit strategy. Candidate genes were chosen according to a bibliographic analysis showing their direct or indirect involvement in the sex differentiation cascade in vertebrates. All the primers were purchased from Eurogentec (Eurogentec, Seraing, Belgium). These primers were manually designed respecting whenever possible the following restriction parameters : 21-23 bp length, no more than 4 identical successive nucleotides, 30-70% GC content, a maximum of 2 G or C among the five 3'-end bases, no primer dimers and a short amplicon size (70-150 bp). Secondary structures were searched with DNA mfold (http://bioinfo.math.rpi.edu/~mfold/DNA/form1.cgi). Whenever possible, each pair was chosen with at least one primer flanking an intron-exon boundary, in order to prevent genomic amplification.	Real-time RT-PCR	Real-time RT-PCR was carried out on an iCycler iQTM (BioRad, Hercules, CA). Reactions were performed in 20µl with 300nM of each primer, 5µl of a 1/50 dilution of the RT reaction and the SYBER-Green PCR master Mix (Eurogentec) according to the manufacturer's instructions. After two incubation steps (50°C 10 min, 95°C 2 min), the thermal cycling protocol was 95°C for 10 min followed by 40 cycles of PCR (95°C 30 s, 60°C or 65°C 1 min). For each primer set the efficiency of the PCR reaction (linear equation : y = slope + intercept) was measured in triplicate on serial dilutions of the same cDNA sample (pool of reverse transcribed RNA samples). Real-time PCR efficiencies for each reaction were then calculated using the formula : Efficiency (E)=[10(1/slope)]-1. Melting curve analysis was also performed for each gene to check the specificity and identity of the RT-PCR products. The relative amount of the target RNA called the Starting Quantity (SQ) was then determined using the I-Cycler IQ software by comparison with the corresponding standard curve for each sample run in duplicate. SQ were calculated as follow : SQ = [10((Ct -intercept)/slope))]-1 where Ct is the Cycle threshold of the unknown sample. Each transcript level was then normalized by division with the expression values of the constitutive elongation factor 1 alpha (EF1a) used as an internal standard.	Female J110 - 1	Oncorhynchus mykiss	GSM38881 extract 1	total RNA	GSM38881 LE 1	1	GSM38881	assay	Rainbow trout sex differentiation cascade-related genes investigated using RT-PCR	P-GSE2151-1	GSM38881_sample_table.txt norm	GSM38881_sample_table.txt	ftp://ftp.ebi.ac.uk/pub/databases/microarray/data/experiment/GEOD/E-GEOD-2151/E-GEOD-2151.processed.1.zip
GSM38880 1	Large-scale temporal gene expression profiling during gonadal differentiation and early gametogenesis in rainbow-trout	Animals and samplings:	Research involving animal experimentation has been approved by the author's institution (authorization number 35-14) and conforms to NIH guidelines. All-male and all-female rainbow trout populations were obtained from the INRA experimental fish farm (Drennec, France) using breeders from the Mirwart strain. At 55 days post-fertilization (55 dpf, i.e., the onset of the free swimming), a male and female batch of 1500 fry each were transferred in 0.3 m3 tanks with recirculating water conditions. They were held at 12°C under constant photoperiod (12h light : 12h dark) and fed ad libitum with a commercial diet (dry pellet food, BiomarTM, Brande, Denmark). In each group, from 20 to 100 gonads, depending on the age of the fish, were sampled and pooled in duplicates at various stages of development : the onset of the free swimming period after complete yolk resumption (Day 0 = D0), D7, D12 (around the first occurrence of oocyte meiosis), D27 (first ovarian lamellar structures), D60 (first previtellogenic oocytes), D90 and D110. There were immediately frozen in liquid nitrogen and stored at -80°C until RNA extraction. Additional gonads were sampled at the same dates for histological analysis performed as previously described.	Total RNA extraction and Reverse Transcription:	Total RNA was extracted using TRIzol reagent (Invitrogen, Cergy Pontoise, France). Total RNA concentration was determined with an Agilent 2100 Bioanalyzer and the RNA 6000 LabChip kit (Agilent Technologies, Stockport, UK) according to the manufacturer's instructions. For cDNA synthesis, 1µg of RNA was denaturated in the presence of random hexamers (0.5µg) for 5 min at 70°C, and then chilled on ice. Reverse transcription (RT) was performed at 37°C for 1 h using M-MLV reverse transcriptase (Promega, Madison, WI) as described by the manufacturer.	Primers design:	Trout candidate gene homologues were searched in international public databanks using a reciprocal blast hit strategy. Candidate genes were chosen according to a bibliographic analysis showing their direct or indirect involvement in the sex differentiation cascade in vertebrates. All the primers were purchased from Eurogentec (Eurogentec, Seraing, Belgium). These primers were manually designed respecting whenever possible the following restriction parameters : 21-23 bp length, no more than 4 identical successive nucleotides, 30-70% GC content, a maximum of 2 G or C among the five 3'-end bases, no primer dimers and a short amplicon size (70-150 bp). Secondary structures were searched with DNA mfold (http://bioinfo.math.rpi.edu/~mfold/DNA/form1.cgi). Whenever possible, each pair was chosen with at least one primer flanking an intron-exon boundary, in order to prevent genomic amplification.	Real-time RT-PCR	Real-time RT-PCR was carried out on an iCycler iQTM (BioRad, Hercules, CA). Reactions were performed in 20µl with 300nM of each primer, 5µl of a 1/50 dilution of the RT reaction and the SYBER-Green PCR master Mix (Eurogentec) according to the manufacturer's instructions. After two incubation steps (50°C 10 min, 95°C 2 min), the thermal cycling protocol was 95°C for 10 min followed by 40 cycles of PCR (95°C 30 s, 60°C or 65°C 1 min). For each primer set the efficiency of the PCR reaction (linear equation : y = slope + intercept) was measured in triplicate on serial dilutions of the same cDNA sample (pool of reverse transcribed RNA samples). Real-time PCR efficiencies for each reaction were then calculated using the formula : Efficiency (E)=[10(1/slope)]-1. Melting curve analysis was also performed for each gene to check the specificity and identity of the RT-PCR products. The relative amount of the target RNA called the Starting Quantity (SQ) was then determined using the I-Cycler IQ software by comparison with the corresponding standard curve for each sample run in duplicate. SQ were calculated as follow : SQ = [10((Ct -intercept)/slope))]-1 where Ct is the Cycle threshold of the unknown sample. Each transcript level was then normalized by division with the expression values of the constitutive elongation factor 1 alpha (EF1a) used as an internal standard.	Female J90 - 2	Oncorhynchus mykiss	GSM38880 extract 1	total RNA	GSM38880 LE 1	1	GSM38880	assay	Rainbow trout sex differentiation cascade-related genes investigated using RT-PCR	P-GSE2151-1	GSM38880_sample_table.txt norm	GSM38880_sample_table.txt	ftp://ftp.ebi.ac.uk/pub/databases/microarray/data/experiment/GEOD/E-GEOD-2151/E-GEOD-2151.processed.1.zip
GSM38879 1	Large-scale temporal gene expression profiling during gonadal differentiation and early gametogenesis in rainbow-trout	Animals and samplings:	Research involving animal experimentation has been approved by the author's institution (authorization number 35-14) and conforms to NIH guidelines. All-male and all-female rainbow trout populations were obtained from the INRA experimental fish farm (Drennec, France) using breeders from the Mirwart strain. At 55 days post-fertilization (55 dpf, i.e., the onset of the free swimming), a male and female batch of 1500 fry each were transferred in 0.3 m3 tanks with recirculating water conditions. They were held at 12°C under constant photoperiod (12h light : 12h dark) and fed ad libitum with a commercial diet (dry pellet food, BiomarTM, Brande, Denmark). In each group, from 20 to 100 gonads, depending on the age of the fish, were sampled and pooled in duplicates at various stages of development : the onset of the free swimming period after complete yolk resumption (Day 0 = D0), D7, D12 (around the first occurrence of oocyte meiosis), D27 (first ovarian lamellar structures), D60 (first previtellogenic oocytes), D90 and D110. There were immediately frozen in liquid nitrogen and stored at -80°C until RNA extraction. Additional gonads were sampled at the same dates for histological analysis performed as previously described.	Total RNA extraction and Reverse Transcription:	Total RNA was extracted using TRIzol reagent (Invitrogen, Cergy Pontoise, France). Total RNA concentration was determined with an Agilent 2100 Bioanalyzer and the RNA 6000 LabChip kit (Agilent Technologies, Stockport, UK) according to the manufacturer's instructions. For cDNA synthesis, 1µg of RNA was denaturated in the presence of random hexamers (0.5µg) for 5 min at 70°C, and then chilled on ice. Reverse transcription (RT) was performed at 37°C for 1 h using M-MLV reverse transcriptase (Promega, Madison, WI) as described by the manufacturer.	Primers design:	Trout candidate gene homologues were searched in international public databanks using a reciprocal blast hit strategy. Candidate genes were chosen according to a bibliographic analysis showing their direct or indirect involvement in the sex differentiation cascade in vertebrates. All the primers were purchased from Eurogentec (Eurogentec, Seraing, Belgium). These primers were manually designed respecting whenever possible the following restriction parameters : 21-23 bp length, no more than 4 identical successive nucleotides, 30-70% GC content, a maximum of 2 G or C among the five 3'-end bases, no primer dimers and a short amplicon size (70-150 bp). Secondary structures were searched with DNA mfold (http://bioinfo.math.rpi.edu/~mfold/DNA/form1.cgi). Whenever possible, each pair was chosen with at least one primer flanking an intron-exon boundary, in order to prevent genomic amplification.	Real-time RT-PCR	Real-time RT-PCR was carried out on an iCycler iQTM (BioRad, Hercules, CA). Reactions were performed in 20µl with 300nM of each primer, 5µl of a 1/50 dilution of the RT reaction and the SYBER-Green PCR master Mix (Eurogentec) according to the manufacturer's instructions. After two incubation steps (50°C 10 min, 95°C 2 min), the thermal cycling protocol was 95°C for 10 min followed by 40 cycles of PCR (95°C 30 s, 60°C or 65°C 1 min). For each primer set the efficiency of the PCR reaction (linear equation : y = slope + intercept) was measured in triplicate on serial dilutions of the same cDNA sample (pool of reverse transcribed RNA samples). Real-time PCR efficiencies for each reaction were then calculated using the formula : Efficiency (E)=[10(1/slope)]-1. Melting curve analysis was also performed for each gene to check the specificity and identity of the RT-PCR products. The relative amount of the target RNA called the Starting Quantity (SQ) was then determined using the I-Cycler IQ software by comparison with the corresponding standard curve for each sample run in duplicate. SQ were calculated as follow : SQ = [10((Ct -intercept)/slope))]-1 where Ct is the Cycle threshold of the unknown sample. Each transcript level was then normalized by division with the expression values of the constitutive elongation factor 1 alpha (EF1a) used as an internal standard.	Female J90 - 1	Oncorhynchus mykiss	GSM38879 extract 1	total RNA	GSM38879 LE 1	1	GSM38879	assay	Rainbow trout sex differentiation cascade-related genes investigated using RT-PCR	P-GSE2151-1	GSM38879_sample_table.txt norm	GSM38879_sample_table.txt	ftp://ftp.ebi.ac.uk/pub/databases/microarray/data/experiment/GEOD/E-GEOD-2151/E-GEOD-2151.processed.1.zip
GSM38878 1	Large-scale temporal gene expression profiling during gonadal differentiation and early gametogenesis in rainbow-trout	Animals and samplings:	Research involving animal experimentation has been approved by the author's institution (authorization number 35-14) and conforms to NIH guidelines. All-male and all-female rainbow trout populations were obtained from the INRA experimental fish farm (Drennec, France) using breeders from the Mirwart strain. At 55 days post-fertilization (55 dpf, i.e., the onset of the free swimming), a male and female batch of 1500 fry each were transferred in 0.3 m3 tanks with recirculating water conditions. They were held at 12°C under constant photoperiod (12h light : 12h dark) and fed ad libitum with a commercial diet (dry pellet food, BiomarTM, Brande, Denmark). In each group, from 20 to 100 gonads, depending on the age of the fish, were sampled and pooled in duplicates at various stages of development : the onset of the free swimming period after complete yolk resumption (Day 0 = D0), D7, D12 (around the first occurrence of oocyte meiosis), D27 (first ovarian lamellar structures), D60 (first previtellogenic oocytes), D90 and D110. There were immediately frozen in liquid nitrogen and stored at -80°C until RNA extraction. Additional gonads were sampled at the same dates for histological analysis performed as previously described.	Total RNA extraction and Reverse Transcription:	Total RNA was extracted using TRIzol reagent (Invitrogen, Cergy Pontoise, France). Total RNA concentration was determined with an Agilent 2100 Bioanalyzer and the RNA 6000 LabChip kit (Agilent Technologies, Stockport, UK) according to the manufacturer's instructions. For cDNA synthesis, 1µg of RNA was denaturated in the presence of random hexamers (0.5µg) for 5 min at 70°C, and then chilled on ice. Reverse transcription (RT) was performed at 37°C for 1 h using M-MLV reverse transcriptase (Promega, Madison, WI) as described by the manufacturer.	Primers design:	Trout candidate gene homologues were searched in international public databanks using a reciprocal blast hit strategy. Candidate genes were chosen according to a bibliographic analysis showing their direct or indirect involvement in the sex differentiation cascade in vertebrates. All the primers were purchased from Eurogentec (Eurogentec, Seraing, Belgium). These primers were manually designed respecting whenever possible the following restriction parameters : 21-23 bp length, no more than 4 identical successive nucleotides, 30-70% GC content, a maximum of 2 G or C among the five 3'-end bases, no primer dimers and a short amplicon size (70-150 bp). Secondary structures were searched with DNA mfold (http://bioinfo.math.rpi.edu/~mfold/DNA/form1.cgi). Whenever possible, each pair was chosen with at least one primer flanking an intron-exon boundary, in order to prevent genomic amplification.	Real-time RT-PCR	Real-time RT-PCR was carried out on an iCycler iQTM (BioRad, Hercules, CA). Reactions were performed in 20µl with 300nM of each primer, 5µl of a 1/50 dilution of the RT reaction and the SYBER-Green PCR master Mix (Eurogentec) according to the manufacturer's instructions. After two incubation steps (50°C 10 min, 95°C 2 min), the thermal cycling protocol was 95°C for 10 min followed by 40 cycles of PCR (95°C 30 s, 60°C or 65°C 1 min). For each primer set the efficiency of the PCR reaction (linear equation : y = slope + intercept) was measured in triplicate on serial dilutions of the same cDNA sample (pool of reverse transcribed RNA samples). Real-time PCR efficiencies for each reaction were then calculated using the formula : Efficiency (E)=[10(1/slope)]-1. Melting curve analysis was also performed for each gene to check the specificity and identity of the RT-PCR products. The relative amount of the target RNA called the Starting Quantity (SQ) was then determined using the I-Cycler IQ software by comparison with the corresponding standard curve for each sample run in duplicate. SQ were calculated as follow : SQ = [10((Ct -intercept)/slope))]-1 where Ct is the Cycle threshold of the unknown sample. Each transcript level was then normalized by division with the expression values of the constitutive elongation factor 1 alpha (EF1a) used as an internal standard.	Female J60 - 2	Oncorhynchus mykiss	GSM38878 extract 1	total RNA	GSM38878 LE 1	1	GSM38878	assay	Rainbow trout sex differentiation cascade-related genes investigated using RT-PCR	P-GSE2151-1	GSM38878_sample_table.txt norm	GSM38878_sample_table.txt	ftp://ftp.ebi.ac.uk/pub/databases/microarray/data/experiment/GEOD/E-GEOD-2151/E-GEOD-2151.processed.1.zip
GSM38877 1	Large-scale temporal gene expression profiling during gonadal differentiation and early gametogenesis in rainbow-trout	Animals and samplings:	Research involving animal experimentation has been approved by the author's institution (authorization number 35-14) and conforms to NIH guidelines. All-male and all-female rainbow trout populations were obtained from the INRA experimental fish farm (Drennec, France) using breeders from the Mirwart strain. At 55 days post-fertilization (55 dpf, i.e., the onset of the free swimming), a male and female batch of 1500 fry each were transferred in 0.3 m3 tanks with recirculating water conditions. They were held at 12°C under constant photoperiod (12h light : 12h dark) and fed ad libitum with a commercial diet (dry pellet food, BiomarTM, Brande, Denmark). In each group, from 20 to 100 gonads, depending on the age of the fish, were sampled and pooled in duplicates at various stages of development : the onset of the free swimming period after complete yolk resumption (Day 0 = D0), D7, D12 (around the first occurrence of oocyte meiosis), D27 (first ovarian lamellar structures), D60 (first previtellogenic oocytes), D90 and D110. There were immediately frozen in liquid nitrogen and stored at -80°C until RNA extraction. Additional gonads were sampled at the same dates for histological analysis performed as previously described.	Total RNA extraction and Reverse Transcription:	Total RNA was extracted using TRIzol reagent (Invitrogen, Cergy Pontoise, France). Total RNA concentration was determined with an Agilent 2100 Bioanalyzer and the RNA 6000 LabChip kit (Agilent Technologies, Stockport, UK) according to the manufacturer's instructions. For cDNA synthesis, 1µg of RNA was denaturated in the presence of random hexamers (0.5µg) for 5 min at 70°C, and then chilled on ice. Reverse transcription (RT) was performed at 37°C for 1 h using M-MLV reverse transcriptase (Promega, Madison, WI) as described by the manufacturer.	Primers design:	Trout candidate gene homologues were searched in international public databanks using a reciprocal blast hit strategy. Candidate genes were chosen according to a bibliographic analysis showing their direct or indirect involvement in the sex differentiation cascade in vertebrates. All the primers were purchased from Eurogentec (Eurogentec, Seraing, Belgium). These primers were manually designed respecting whenever possible the following restriction parameters : 21-23 bp length, no more than 4 identical successive nucleotides, 30-70% GC content, a maximum of 2 G or C among the five 3'-end bases, no primer dimers and a short amplicon size (70-150 bp). Secondary structures were searched with DNA mfold (http://bioinfo.math.rpi.edu/~mfold/DNA/form1.cgi). Whenever possible, each pair was chosen with at least one primer flanking an intron-exon boundary, in order to prevent genomic amplification.	Real-time RT-PCR	Real-time RT-PCR was carried out on an iCycler iQTM (BioRad, Hercules, CA). Reactions were performed in 20µl with 300nM of each primer, 5µl of a 1/50 dilution of the RT reaction and the SYBER-Green PCR master Mix (Eurogentec) according to the manufacturer's instructions. After two incubation steps (50°C 10 min, 95°C 2 min), the thermal cycling protocol was 95°C for 10 min followed by 40 cycles of PCR (95°C 30 s, 60°C or 65°C 1 min). For each primer set the efficiency of the PCR reaction (linear equation : y = slope + intercept) was measured in triplicate on serial dilutions of the same cDNA sample (pool of reverse transcribed RNA samples). Real-time PCR efficiencies for each reaction were then calculated using the formula : Efficiency (E)=[10(1/slope)]-1. Melting curve analysis was also performed for each gene to check the specificity and identity of the RT-PCR products. The relative amount of the target RNA called the Starting Quantity (SQ) was then determined using the I-Cycler IQ software by comparison with the corresponding standard curve for each sample run in duplicate. SQ were calculated as follow : SQ = [10((Ct -intercept)/slope))]-1 where Ct is the Cycle threshold of the unknown sample. Each transcript level was then normalized by division with the expression values of the constitutive elongation factor 1 alpha (EF1a) used as an internal standard.	Female J60 - 1	Oncorhynchus mykiss	GSM38877 extract 1	total RNA	GSM38877 LE 1	1	GSM38877	assay	Rainbow trout sex differentiation cascade-related genes investigated using RT-PCR	P-GSE2151-1	GSM38877_sample_table.txt norm	GSM38877_sample_table.txt	ftp://ftp.ebi.ac.uk/pub/databases/microarray/data/experiment/GEOD/E-GEOD-2151/E-GEOD-2151.processed.1.zip
GSM38876 1	Large-scale temporal gene expression profiling during gonadal differentiation and early gametogenesis in rainbow-trout	Animals and samplings:	Research involving animal experimentation has been approved by the author's institution (authorization number 35-14) and conforms to NIH guidelines. All-male and all-female rainbow trout populations were obtained from the INRA experimental fish farm (Drennec, France) using breeders from the Mirwart strain. At 55 days post-fertilization (55 dpf, i.e., the onset of the free swimming), a male and female batch of 1500 fry each were transferred in 0.3 m3 tanks with recirculating water conditions. They were held at 12°C under constant photoperiod (12h light : 12h dark) and fed ad libitum with a commercial diet (dry pellet food, BiomarTM, Brande, Denmark). In each group, from 20 to 100 gonads, depending on the age of the fish, were sampled and pooled in duplicates at various stages of development : the onset of the free swimming period after complete yolk resumption (Day 0 = D0), D7, D12 (around the first occurrence of oocyte meiosis), D27 (first ovarian lamellar structures), D60 (first previtellogenic oocytes), D90 and D110. There were immediately frozen in liquid nitrogen and stored at -80°C until RNA extraction. Additional gonads were sampled at the same dates for histological analysis performed as previously described.	Total RNA extraction and Reverse Transcription:	Total RNA was extracted using TRIzol reagent (Invitrogen, Cergy Pontoise, France). Total RNA concentration was determined with an Agilent 2100 Bioanalyzer and the RNA 6000 LabChip kit (Agilent Technologies, Stockport, UK) according to the manufacturer's instructions. For cDNA synthesis, 1µg of RNA was denaturated in the presence of random hexamers (0.5µg) for 5 min at 70°C, and then chilled on ice. Reverse transcription (RT) was performed at 37°C for 1 h using M-MLV reverse transcriptase (Promega, Madison, WI) as described by the manufacturer.	Primers design:	Trout candidate gene homologues were searched in international public databanks using a reciprocal blast hit strategy. Candidate genes were chosen according to a bibliographic analysis showing their direct or indirect involvement in the sex differentiation cascade in vertebrates. All the primers were purchased from Eurogentec (Eurogentec, Seraing, Belgium). These primers were manually designed respecting whenever possible the following restriction parameters : 21-23 bp length, no more than 4 identical successive nucleotides, 30-70% GC content, a maximum of 2 G or C among the five 3'-end bases, no primer dimers and a short amplicon size (70-150 bp). Secondary structures were searched with DNA mfold (http://bioinfo.math.rpi.edu/~mfold/DNA/form1.cgi). Whenever possible, each pair was chosen with at least one primer flanking an intron-exon boundary, in order to prevent genomic amplification.	Real-time RT-PCR	Real-time RT-PCR was carried out on an iCycler iQTM (BioRad, Hercules, CA). Reactions were performed in 20µl with 300nM of each primer, 5µl of a 1/50 dilution of the RT reaction and the SYBER-Green PCR master Mix (Eurogentec) according to the manufacturer's instructions. After two incubation steps (50°C 10 min, 95°C 2 min), the thermal cycling protocol was 95°C for 10 min followed by 40 cycles of PCR (95°C 30 s, 60°C or 65°C 1 min). For each primer set the efficiency of the PCR reaction (linear equation : y = slope + intercept) was measured in triplicate on serial dilutions of the same cDNA sample (pool of reverse transcribed RNA samples). Real-time PCR efficiencies for each reaction were then calculated using the formula : Efficiency (E)=[10(1/slope)]-1. Melting curve analysis was also performed for each gene to check the specificity and identity of the RT-PCR products. The relative amount of the target RNA called the Starting Quantity (SQ) was then determined using the I-Cycler IQ software by comparison with the corresponding standard curve for each sample run in duplicate. SQ were calculated as follow : SQ = [10((Ct -intercept)/slope))]-1 where Ct is the Cycle threshold of the unknown sample. Each transcript level was then normalized by division with the expression values of the constitutive elongation factor 1 alpha (EF1a) used as an internal standard.	Female J27 - 2	Oncorhynchus mykiss	GSM38876 extract 1	total RNA	GSM38876 LE 1	1	GSM38876	assay	Rainbow trout sex differentiation cascade-related genes investigated using RT-PCR	P-GSE2151-1	GSM38876_sample_table.txt norm	GSM38876_sample_table.txt	ftp://ftp.ebi.ac.uk/pub/databases/microarray/data/experiment/GEOD/E-GEOD-2151/E-GEOD-2151.processed.1.zip
GSM38875 1	Large-scale temporal gene expression profiling during gonadal differentiation and early gametogenesis in rainbow-trout	Animals and samplings:	Research involving animal experimentation has been approved by the author's institution (authorization number 35-14) and conforms to NIH guidelines. All-male and all-female rainbow trout populations were obtained from the INRA experimental fish farm (Drennec, France) using breeders from the Mirwart strain. At 55 days post-fertilization (55 dpf, i.e., the onset of the free swimming), a male and female batch of 1500 fry each were transferred in 0.3 m3 tanks with recirculating water conditions. They were held at 12°C under constant photoperiod (12h light : 12h dark) and fed ad libitum with a commercial diet (dry pellet food, BiomarTM, Brande, Denmark). In each group, from 20 to 100 gonads, depending on the age of the fish, were sampled and pooled in duplicates at various stages of development : the onset of the free swimming period after complete yolk resumption (Day 0 = D0), D7, D12 (around the first occurrence of oocyte meiosis), D27 (first ovarian lamellar structures), D60 (first previtellogenic oocytes), D90 and D110. There were immediately frozen in liquid nitrogen and stored at -80°C until RNA extraction. Additional gonads were sampled at the same dates for histological analysis performed as previously described.	Total RNA extraction and Reverse Transcription:	Total RNA was extracted using TRIzol reagent (Invitrogen, Cergy Pontoise, France). Total RNA concentration was determined with an Agilent 2100 Bioanalyzer and the RNA 6000 LabChip kit (Agilent Technologies, Stockport, UK) according to the manufacturer's instructions. For cDNA synthesis, 1µg of RNA was denaturated in the presence of random hexamers (0.5µg) for 5 min at 70°C, and then chilled on ice. Reverse transcription (RT) was performed at 37°C for 1 h using M-MLV reverse transcriptase (Promega, Madison, WI) as described by the manufacturer.	Primers design:	Trout candidate gene homologues were searched in international public databanks using a reciprocal blast hit strategy. Candidate genes were chosen according to a bibliographic analysis showing their direct or indirect involvement in the sex differentiation cascade in vertebrates. All the primers were purchased from Eurogentec (Eurogentec, Seraing, Belgium). These primers were manually designed respecting whenever possible the following restriction parameters : 21-23 bp length, no more than 4 identical successive nucleotides, 30-70% GC content, a maximum of 2 G or C among the five 3'-end bases, no primer dimers and a short amplicon size (70-150 bp). Secondary structures were searched with DNA mfold (http://bioinfo.math.rpi.edu/~mfold/DNA/form1.cgi). Whenever possible, each pair was chosen with at least one primer flanking an intron-exon boundary, in order to prevent genomic amplification.	Real-time RT-PCR	Real-time RT-PCR was carried out on an iCycler iQTM (BioRad, Hercules, CA). Reactions were performed in 20µl with 300nM of each primer, 5µl of a 1/50 dilution of the RT reaction and the SYBER-Green PCR master Mix (Eurogentec) according to the manufacturer's instructions. After two incubation steps (50°C 10 min, 95°C 2 min), the thermal cycling protocol was 95°C for 10 min followed by 40 cycles of PCR (95°C 30 s, 60°C or 65°C 1 min). For each primer set the efficiency of the PCR reaction (linear equation : y = slope + intercept) was measured in triplicate on serial dilutions of the same cDNA sample (pool of reverse transcribed RNA samples). Real-time PCR efficiencies for each reaction were then calculated using the formula : Efficiency (E)=[10(1/slope)]-1. Melting curve analysis was also performed for each gene to check the specificity and identity of the RT-PCR products. The relative amount of the target RNA called the Starting Quantity (SQ) was then determined using the I-Cycler IQ software by comparison with the corresponding standard curve for each sample run in duplicate. SQ were calculated as follow : SQ = [10((Ct -intercept)/slope))]-1 where Ct is the Cycle threshold of the unknown sample. Each transcript level was then normalized by division with the expression values of the constitutive elongation factor 1 alpha (EF1a) used as an internal standard.	Female J27 - 1	Oncorhynchus mykiss	GSM38875 extract 1	total RNA	GSM38875 LE 1	1	GSM38875	assay	Rainbow trout sex differentiation cascade-related genes investigated using RT-PCR	P-GSE2151-1	GSM38875_sample_table.txt norm	GSM38875_sample_table.txt	ftp://ftp.ebi.ac.uk/pub/databases/microarray/data/experiment/GEOD/E-GEOD-2151/E-GEOD-2151.processed.1.zip
GSM38874 1	Large-scale temporal gene expression profiling during gonadal differentiation and early gametogenesis in rainbow-trout	Animals and samplings:	Research involving animal experimentation has been approved by the author's institution (authorization number 35-14) and conforms to NIH guidelines. All-male and all-female rainbow trout populations were obtained from the INRA experimental fish farm (Drennec, France) using breeders from the Mirwart strain. At 55 days post-fertilization (55 dpf, i.e., the onset of the free swimming), a male and female batch of 1500 fry each were transferred in 0.3 m3 tanks with recirculating water conditions. They were held at 12°C under constant photoperiod (12h light : 12h dark) and fed ad libitum with a commercial diet (dry pellet food, BiomarTM, Brande, Denmark). In each group, from 20 to 100 gonads, depending on the age of the fish, were sampled and pooled in duplicates at various stages of development : the onset of the free swimming period after complete yolk resumption (Day 0 = D0), D7, D12 (around the first occurrence of oocyte meiosis), D27 (first ovarian lamellar structures), D60 (first previtellogenic oocytes), D90 and D110. There were immediately frozen in liquid nitrogen and stored at -80°C until RNA extraction. Additional gonads were sampled at the same dates for histological analysis performed as previously described.	Total RNA extraction and Reverse Transcription:	Total RNA was extracted using TRIzol reagent (Invitrogen, Cergy Pontoise, France). Total RNA concentration was determined with an Agilent 2100 Bioanalyzer and the RNA 6000 LabChip kit (Agilent Technologies, Stockport, UK) according to the manufacturer's instructions. For cDNA synthesis, 1µg of RNA was denaturated in the presence of random hexamers (0.5µg) for 5 min at 70°C, and then chilled on ice. Reverse transcription (RT) was performed at 37°C for 1 h using M-MLV reverse transcriptase (Promega, Madison, WI) as described by the manufacturer.	Primers design:	Trout candidate gene homologues were searched in international public databanks using a reciprocal blast hit strategy. Candidate genes were chosen according to a bibliographic analysis showing their direct or indirect involvement in the sex differentiation cascade in vertebrates. All the primers were purchased from Eurogentec (Eurogentec, Seraing, Belgium). These primers were manually designed respecting whenever possible the following restriction parameters : 21-23 bp length, no more than 4 identical successive nucleotides, 30-70% GC content, a maximum of 2 G or C among the five 3'-end bases, no primer dimers and a short amplicon size (70-150 bp). Secondary structures were searched with DNA mfold (http://bioinfo.math.rpi.edu/~mfold/DNA/form1.cgi). Whenever possible, each pair was chosen with at least one primer flanking an intron-exon boundary, in order to prevent genomic amplification.	Real-time RT-PCR	Real-time RT-PCR was carried out on an iCycler iQTM (BioRad, Hercules, CA). Reactions were performed in 20µl with 300nM of each primer, 5µl of a 1/50 dilution of the RT reaction and the SYBER-Green PCR master Mix (Eurogentec) according to the manufacturer's instructions. After two incubation steps (50°C 10 min, 95°C 2 min), the thermal cycling protocol was 95°C for 10 min followed by 40 cycles of PCR (95°C 30 s, 60°C or 65°C 1 min). For each primer set the efficiency of the PCR reaction (linear equation : y = slope + intercept) was measured in triplicate on serial dilutions of the same cDNA sample (pool of reverse transcribed RNA samples). Real-time PCR efficiencies for each reaction were then calculated using the formula : Efficiency (E)=[10(1/slope)]-1. Melting curve analysis was also performed for each gene to check the specificity and identity of the RT-PCR products. The relative amount of the target RNA called the Starting Quantity (SQ) was then determined using the I-Cycler IQ software by comparison with the corresponding standard curve for each sample run in duplicate. SQ were calculated as follow : SQ = [10((Ct -intercept)/slope))]-1 where Ct is the Cycle threshold of the unknown sample. Each transcript level was then normalized by division with the expression values of the constitutive elongation factor 1 alpha (EF1a) used as an internal standard.	Female J12 - 2	Oncorhynchus mykiss	GSM38874 extract 1	total RNA	GSM38874 LE 1	1	GSM38874	assay	Rainbow trout sex differentiation cascade-related genes investigated using RT-PCR	P-GSE2151-1	GSM38874_sample_table.txt norm	GSM38874_sample_table.txt	ftp://ftp.ebi.ac.uk/pub/databases/microarray/data/experiment/GEOD/E-GEOD-2151/E-GEOD-2151.processed.1.zip
GSM38873 1	Large-scale temporal gene expression profiling during gonadal differentiation and early gametogenesis in rainbow-trout	Animals and samplings:	Research involving animal experimentation has been approved by the author's institution (authorization number 35-14) and conforms to NIH guidelines. All-male and all-female rainbow trout populations were obtained from the INRA experimental fish farm (Drennec, France) using breeders from the Mirwart strain. At 55 days post-fertilization (55 dpf, i.e., the onset of the free swimming), a male and female batch of 1500 fry each were transferred in 0.3 m3 tanks with recirculating water conditions. They were held at 12°C under constant photoperiod (12h light : 12h dark) and fed ad libitum with a commercial diet (dry pellet food, BiomarTM, Brande, Denmark). In each group, from 20 to 100 gonads, depending on the age of the fish, were sampled and pooled in duplicates at various stages of development : the onset of the free swimming period after complete yolk resumption (Day 0 = D0), D7, D12 (around the first occurrence of oocyte meiosis), D27 (first ovarian lamellar structures), D60 (first previtellogenic oocytes), D90 and D110. There were immediately frozen in liquid nitrogen and stored at -80°C until RNA extraction. Additional gonads were sampled at the same dates for histological analysis performed as previously described.	Total RNA extraction and Reverse Transcription:	Total RNA was extracted using TRIzol reagent (Invitrogen, Cergy Pontoise, France). Total RNA concentration was determined with an Agilent 2100 Bioanalyzer and the RNA 6000 LabChip kit (Agilent Technologies, Stockport, UK) according to the manufacturer's instructions. For cDNA synthesis, 1µg of RNA was denaturated in the presence of random hexamers (0.5µg) for 5 min at 70°C, and then chilled on ice. Reverse transcription (RT) was performed at 37°C for 1 h using M-MLV reverse transcriptase (Promega, Madison, WI) as described by the manufacturer.	Primers design:	Trout candidate gene homologues were searched in international public databanks using a reciprocal blast hit strategy. Candidate genes were chosen according to a bibliographic analysis showing their direct or indirect involvement in the sex differentiation cascade in vertebrates. All the primers were purchased from Eurogentec (Eurogentec, Seraing, Belgium). These primers were manually designed respecting whenever possible the following restriction parameters : 21-23 bp length, no more than 4 identical successive nucleotides, 30-70% GC content, a maximum of 2 G or C among the five 3'-end bases, no primer dimers and a short amplicon size (70-150 bp). Secondary structures were searched with DNA mfold (http://bioinfo.math.rpi.edu/~mfold/DNA/form1.cgi). Whenever possible, each pair was chosen with at least one primer flanking an intron-exon boundary, in order to prevent genomic amplification.	Real-time RT-PCR	Real-time RT-PCR was carried out on an iCycler iQTM (BioRad, Hercules, CA). Reactions were performed in 20µl with 300nM of each primer, 5µl of a 1/50 dilution of the RT reaction and the SYBER-Green PCR master Mix (Eurogentec) according to the manufacturer's instructions. After two incubation steps (50°C 10 min, 95°C 2 min), the thermal cycling protocol was 95°C for 10 min followed by 40 cycles of PCR (95°C 30 s, 60°C or 65°C 1 min). For each primer set the efficiency of the PCR reaction (linear equation : y = slope + intercept) was measured in triplicate on serial dilutions of the same cDNA sample (pool of reverse transcribed RNA samples). Real-time PCR efficiencies for each reaction were then calculated using the formula : Efficiency (E)=[10(1/slope)]-1. Melting curve analysis was also performed for each gene to check the specificity and identity of the RT-PCR products. The relative amount of the target RNA called the Starting Quantity (SQ) was then determined using the I-Cycler IQ software by comparison with the corresponding standard curve for each sample run in duplicate. SQ were calculated as follow : SQ = [10((Ct -intercept)/slope))]-1 where Ct is the Cycle threshold of the unknown sample. Each transcript level was then normalized by division with the expression values of the constitutive elongation factor 1 alpha (EF1a) used as an internal standard.	Female J12 - 1	Oncorhynchus mykiss	GSM38873 extract 1	total RNA	GSM38873 LE 1	1	GSM38873	assay	Rainbow trout sex differentiation cascade-related genes investigated using RT-PCR	P-GSE2151-1	GSM38873_sample_table.txt norm	GSM38873_sample_table.txt	ftp://ftp.ebi.ac.uk/pub/databases/microarray/data/experiment/GEOD/E-GEOD-2151/E-GEOD-2151.processed.1.zip
GSM38872 1	Large-scale temporal gene expression profiling during gonadal differentiation and early gametogenesis in rainbow-trout	Animals and samplings:	Research involving animal experimentation has been approved by the author's institution (authorization number 35-14) and conforms to NIH guidelines. All-male and all-female rainbow trout populations were obtained from the INRA experimental fish farm (Drennec, France) using breeders from the Mirwart strain. At 55 days post-fertilization (55 dpf, i.e., the onset of the free swimming), a male and female batch of 1500 fry each were transferred in 0.3 m3 tanks with recirculating water conditions. They were held at 12°C under constant photoperiod (12h light : 12h dark) and fed ad libitum with a commercial diet (dry pellet food, BiomarTM, Brande, Denmark). In each group, from 20 to 100 gonads, depending on the age of the fish, were sampled and pooled in duplicates at various stages of development : the onset of the free swimming period after complete yolk resumption (Day 0 = D0), D7, D12 (around the first occurrence of oocyte meiosis), D27 (first ovarian lamellar structures), D60 (first previtellogenic oocytes), D90 and D110. There were immediately frozen in liquid nitrogen and stored at -80°C until RNA extraction. Additional gonads were sampled at the same dates for histological analysis performed as previously described.	Total RNA extraction and Reverse Transcription:	Total RNA was extracted using TRIzol reagent (Invitrogen, Cergy Pontoise, France). Total RNA concentration was determined with an Agilent 2100 Bioanalyzer and the RNA 6000 LabChip kit (Agilent Technologies, Stockport, UK) according to the manufacturer's instructions. For cDNA synthesis, 1µg of RNA was denaturated in the presence of random hexamers (0.5µg) for 5 min at 70°C, and then chilled on ice. Reverse transcription (RT) was performed at 37°C for 1 h using M-MLV reverse transcriptase (Promega, Madison, WI) as described by the manufacturer.	Primers design:	Trout candidate gene homologues were searched in international public databanks using a reciprocal blast hit strategy. Candidate genes were chosen according to a bibliographic analysis showing their direct or indirect involvement in the sex differentiation cascade in vertebrates. All the primers were purchased from Eurogentec (Eurogentec, Seraing, Belgium). These primers were manually designed respecting whenever possible the following restriction parameters : 21-23 bp length, no more than 4 identical successive nucleotides, 30-70% GC content, a maximum of 2 G or C among the five 3'-end bases, no primer dimers and a short amplicon size (70-150 bp). Secondary structures were searched with DNA mfold (http://bioinfo.math.rpi.edu/~mfold/DNA/form1.cgi). Whenever possible, each pair was chosen with at least one primer flanking an intron-exon boundary, in order to prevent genomic amplification.	Real-time RT-PCR	Real-time RT-PCR was carried out on an iCycler iQTM (BioRad, Hercules, CA). Reactions were performed in 20µl with 300nM of each primer, 5µl of a 1/50 dilution of the RT reaction and the SYBER-Green PCR master Mix (Eurogentec) according to the manufacturer's instructions. After two incubation steps (50°C 10 min, 95°C 2 min), the thermal cycling protocol was 95°C for 10 min followed by 40 cycles of PCR (95°C 30 s, 60°C or 65°C 1 min). For each primer set the efficiency of the PCR reaction (linear equation : y = slope + intercept) was measured in triplicate on serial dilutions of the same cDNA sample (pool of reverse transcribed RNA samples). Real-time PCR efficiencies for each reaction were then calculated using the formula : Efficiency (E)=[10(1/slope)]-1. Melting curve analysis was also performed for each gene to check the specificity and identity of the RT-PCR products. The relative amount of the target RNA called the Starting Quantity (SQ) was then determined using the I-Cycler IQ software by comparison with the corresponding standard curve for each sample run in duplicate. SQ were calculated as follow : SQ = [10((Ct -intercept)/slope))]-1 where Ct is the Cycle threshold of the unknown sample. Each transcript level was then normalized by division with the expression values of the constitutive elongation factor 1 alpha (EF1a) used as an internal standard.	Female J7 - 2	Oncorhynchus mykiss	GSM38872 extract 1	total RNA	GSM38872 LE 1	1	GSM38872	assay	Rainbow trout sex differentiation cascade-related genes investigated using RT-PCR	P-GSE2151-1	GSM38872_sample_table.txt norm	GSM38872_sample_table.txt	ftp://ftp.ebi.ac.uk/pub/databases/microarray/data/experiment/GEOD/E-GEOD-2151/E-GEOD-2151.processed.1.zip
GSM38871 1	Large-scale temporal gene expression profiling during gonadal differentiation and early gametogenesis in rainbow-trout	Animals and samplings:	Research involving animal experimentation has been approved by the author's institution (authorization number 35-14) and conforms to NIH guidelines. All-male and all-female rainbow trout populations were obtained from the INRA experimental fish farm (Drennec, France) using breeders from the Mirwart strain. At 55 days post-fertilization (55 dpf, i.e., the onset of the free swimming), a male and female batch of 1500 fry each were transferred in 0.3 m3 tanks with recirculating water conditions. They were held at 12°C under constant photoperiod (12h light : 12h dark) and fed ad libitum with a commercial diet (dry pellet food, BiomarTM, Brande, Denmark). In each group, from 20 to 100 gonads, depending on the age of the fish, were sampled and pooled in duplicates at various stages of development : the onset of the free swimming period after complete yolk resumption (Day 0 = D0), D7, D12 (around the first occurrence of oocyte meiosis), D27 (first ovarian lamellar structures), D60 (first previtellogenic oocytes), D90 and D110. There were immediately frozen in liquid nitrogen and stored at -80°C until RNA extraction. Additional gonads were sampled at the same dates for histological analysis performed as previously described.	Total RNA extraction and Reverse Transcription:	Total RNA was extracted using TRIzol reagent (Invitrogen, Cergy Pontoise, France). Total RNA concentration was determined with an Agilent 2100 Bioanalyzer and the RNA 6000 LabChip kit (Agilent Technologies, Stockport, UK) according to the manufacturer's instructions. For cDNA synthesis, 1µg of RNA was denaturated in the presence of random hexamers (0.5µg) for 5 min at 70°C, and then chilled on ice. Reverse transcription (RT) was performed at 37°C for 1 h using M-MLV reverse transcriptase (Promega, Madison, WI) as described by the manufacturer.	Primers design:	Trout candidate gene homologues were searched in international public databanks using a reciprocal blast hit strategy. Candidate genes were chosen according to a bibliographic analysis showing their direct or indirect involvement in the sex differentiation cascade in vertebrates. All the primers were purchased from Eurogentec (Eurogentec, Seraing, Belgium). These primers were manually designed respecting whenever possible the following restriction parameters : 21-23 bp length, no more than 4 identical successive nucleotides, 30-70% GC content, a maximum of 2 G or C among the five 3'-end bases, no primer dimers and a short amplicon size (70-150 bp). Secondary structures were searched with DNA mfold (http://bioinfo.math.rpi.edu/~mfold/DNA/form1.cgi). Whenever possible, each pair was chosen with at least one primer flanking an intron-exon boundary, in order to prevent genomic amplification.	Real-time RT-PCR	Real-time RT-PCR was carried out on an iCycler iQTM (BioRad, Hercules, CA). Reactions were performed in 20µl with 300nM of each primer, 5µl of a 1/50 dilution of the RT reaction and the SYBER-Green PCR master Mix (Eurogentec) according to the manufacturer's instructions. After two incubation steps (50°C 10 min, 95°C 2 min), the thermal cycling protocol was 95°C for 10 min followed by 40 cycles of PCR (95°C 30 s, 60°C or 65°C 1 min). For each primer set the efficiency of the PCR reaction (linear equation : y = slope + intercept) was measured in triplicate on serial dilutions of the same cDNA sample (pool of reverse transcribed RNA samples). Real-time PCR efficiencies for each reaction were then calculated using the formula : Efficiency (E)=[10(1/slope)]-1. Melting curve analysis was also performed for each gene to check the specificity and identity of the RT-PCR products. The relative amount of the target RNA called the Starting Quantity (SQ) was then determined using the I-Cycler IQ software by comparison with the corresponding standard curve for each sample run in duplicate. SQ were calculated as follow : SQ = [10((Ct -intercept)/slope))]-1 where Ct is the Cycle threshold of the unknown sample. Each transcript level was then normalized by division with the expression values of the constitutive elongation factor 1 alpha (EF1a) used as an internal standard.	Female J7 - 1	Oncorhynchus mykiss	GSM38871 extract 1	total RNA	GSM38871 LE 1	1	GSM38871	assay	Rainbow trout sex differentiation cascade-related genes investigated using RT-PCR	P-GSE2151-1	GSM38871_sample_table.txt norm	GSM38871_sample_table.txt	ftp://ftp.ebi.ac.uk/pub/databases/microarray/data/experiment/GEOD/E-GEOD-2151/E-GEOD-2151.processed.1.zip
GSM38870 1	Large-scale temporal gene expression profiling during gonadal differentiation and early gametogenesis in rainbow-trout	Animals and samplings:	Research involving animal experimentation has been approved by the author's institution (authorization number 35-14) and conforms to NIH guidelines. All-male and all-female rainbow trout populations were obtained from the INRA experimental fish farm (Drennec, France) using breeders from the Mirwart strain. At 55 days post-fertilization (55 dpf, i.e., the onset of the free swimming), a male and female batch of 1500 fry each were transferred in 0.3 m3 tanks with recirculating water conditions. They were held at 12°C under constant photoperiod (12h light : 12h dark) and fed ad libitum with a commercial diet (dry pellet food, BiomarTM, Brande, Denmark). In each group, from 20 to 100 gonads, depending on the age of the fish, were sampled and pooled in duplicates at various stages of development : the onset of the free swimming period after complete yolk resumption (Day 0 = D0), D7, D12 (around the first occurrence of oocyte meiosis), D27 (first ovarian lamellar structures), D60 (first previtellogenic oocytes), D90 and D110. There were immediately frozen in liquid nitrogen and stored at -80°C until RNA extraction. Additional gonads were sampled at the same dates for histological analysis performed as previously described.	Total RNA extraction and Reverse Transcription:	Total RNA was extracted using TRIzol reagent (Invitrogen, Cergy Pontoise, France). Total RNA concentration was determined with an Agilent 2100 Bioanalyzer and the RNA 6000 LabChip kit (Agilent Technologies, Stockport, UK) according to the manufacturer's instructions. For cDNA synthesis, 1µg of RNA was denaturated in the presence of random hexamers (0.5µg) for 5 min at 70°C, and then chilled on ice. Reverse transcription (RT) was performed at 37°C for 1 h using M-MLV reverse transcriptase (Promega, Madison, WI) as described by the manufacturer.	Primers design:	Trout candidate gene homologues were searched in international public databanks using a reciprocal blast hit strategy. Candidate genes were chosen according to a bibliographic analysis showing their direct or indirect involvement in the sex differentiation cascade in vertebrates. All the primers were purchased from Eurogentec (Eurogentec, Seraing, Belgium). These primers were manually designed respecting whenever possible the following restriction parameters : 21-23 bp length, no more than 4 identical successive nucleotides, 30-70% GC content, a maximum of 2 G or C among the five 3'-end bases, no primer dimers and a short amplicon size (70-150 bp). Secondary structures were searched with DNA mfold (http://bioinfo.math.rpi.edu/~mfold/DNA/form1.cgi). Whenever possible, each pair was chosen with at least one primer flanking an intron-exon boundary, in order to prevent genomic amplification.	Real-time RT-PCR	Real-time RT-PCR was carried out on an iCycler iQTM (BioRad, Hercules, CA). Reactions were performed in 20µl with 300nM of each primer, 5µl of a 1/50 dilution of the RT reaction and the SYBER-Green PCR master Mix (Eurogentec) according to the manufacturer's instructions. After two incubation steps (50°C 10 min, 95°C 2 min), the thermal cycling protocol was 95°C for 10 min followed by 40 cycles of PCR (95°C 30 s, 60°C or 65°C 1 min). For each primer set the efficiency of the PCR reaction (linear equation : y = slope + intercept) was measured in triplicate on serial dilutions of the same cDNA sample (pool of reverse transcribed RNA samples). Real-time PCR efficiencies for each reaction were then calculated using the formula : Efficiency (E)=[10(1/slope)]-1. Melting curve analysis was also performed for each gene to check the specificity and identity of the RT-PCR products. The relative amount of the target RNA called the Starting Quantity (SQ) was then determined using the I-Cycler IQ software by comparison with the corresponding standard curve for each sample run in duplicate. SQ were calculated as follow : SQ = [10((Ct -intercept)/slope))]-1 where Ct is the Cycle threshold of the unknown sample. Each transcript level was then normalized by division with the expression values of the constitutive elongation factor 1 alpha (EF1a) used as an internal standard.	Female J0 - 2	Oncorhynchus mykiss	GSM38870 extract 1	total RNA	GSM38870 LE 1	1	GSM38870	assay	Rainbow trout sex differentiation cascade-related genes investigated using RT-PCR	P-GSE2151-1	GSM38870_sample_table.txt norm	GSM38870_sample_table.txt	ftp://ftp.ebi.ac.uk/pub/databases/microarray/data/experiment/GEOD/E-GEOD-2151/E-GEOD-2151.processed.1.zip
GSM38869 1	Large-scale temporal gene expression profiling during gonadal differentiation and early gametogenesis in rainbow-trout	Animals and samplings:	Research involving animal experimentation has been approved by the author's institution (authorization number 35-14) and conforms to NIH guidelines. All-male and all-female rainbow trout populations were obtained from the INRA experimental fish farm (Drennec, France) using breeders from the Mirwart strain. At 55 days post-fertilization (55 dpf, i.e., the onset of the free swimming), a male and female batch of 1500 fry each were transferred in 0.3 m3 tanks with recirculating water conditions. They were held at 12°C under constant photoperiod (12h light : 12h dark) and fed ad libitum with a commercial diet (dry pellet food, BiomarTM, Brande, Denmark). In each group, from 20 to 100 gonads, depending on the age of the fish, were sampled and pooled in duplicates at various stages of development : the onset of the free swimming period after complete yolk resumption (Day 0 = D0), D7, D12 (around the first occurrence of oocyte meiosis), D27 (first ovarian lamellar structures), D60 (first previtellogenic oocytes), D90 and D110. There were immediately frozen in liquid nitrogen and stored at -80°C until RNA extraction. Additional gonads were sampled at the same dates for histological analysis performed as previously described.	Total RNA extraction and Reverse Transcription:	Total RNA was extracted using TRIzol reagent (Invitrogen, Cergy Pontoise, France). Total RNA concentration was determined with an Agilent 2100 Bioanalyzer and the RNA 6000 LabChip kit (Agilent Technologies, Stockport, UK) according to the manufacturer's instructions. For cDNA synthesis, 1µg of RNA was denaturated in the presence of random hexamers (0.5µg) for 5 min at 70°C, and then chilled on ice. Reverse transcription (RT) was performed at 37°C for 1 h using M-MLV reverse transcriptase (Promega, Madison, WI) as described by the manufacturer.	Primers design:	Trout candidate gene homologues were searched in international public databanks using a reciprocal blast hit strategy. Candidate genes were chosen according to a bibliographic analysis showing their direct or indirect involvement in the sex differentiation cascade in vertebrates. All the primers were purchased from Eurogentec (Eurogentec, Seraing, Belgium). These primers were manually designed respecting whenever possible the following restriction parameters : 21-23 bp length, no more than 4 identical successive nucleotides, 30-70% GC content, a maximum of 2 G or C among the five 3'-end bases, no primer dimers and a short amplicon size (70-150 bp). Secondary structures were searched with DNA mfold (http://bioinfo.math.rpi.edu/~mfold/DNA/form1.cgi). Whenever possible, each pair was chosen with at least one primer flanking an intron-exon boundary, in order to prevent genomic amplification.	Real-time RT-PCR	Real-time RT-PCR was carried out on an iCycler iQTM (BioRad, Hercules, CA). Reactions were performed in 20µl with 300nM of each primer, 5µl of a 1/50 dilution of the RT reaction and the SYBER-Green PCR master Mix (Eurogentec) according to the manufacturer's instructions. After two incubation steps (50°C 10 min, 95°C 2 min), the thermal cycling protocol was 95°C for 10 min followed by 40 cycles of PCR (95°C 30 s, 60°C or 65°C 1 min). For each primer set the efficiency of the PCR reaction (linear equation : y = slope + intercept) was measured in triplicate on serial dilutions of the same cDNA sample (pool of reverse transcribed RNA samples). Real-time PCR efficiencies for each reaction were then calculated using the formula : Efficiency (E)=[10(1/slope)]-1. Melting curve analysis was also performed for each gene to check the specificity and identity of the RT-PCR products. The relative amount of the target RNA called the Starting Quantity (SQ) was then determined using the I-Cycler IQ software by comparison with the corresponding standard curve for each sample run in duplicate. SQ were calculated as follow : SQ = [10((Ct -intercept)/slope))]-1 where Ct is the Cycle threshold of the unknown sample. Each transcript level was then normalized by division with the expression values of the constitutive elongation factor 1 alpha (EF1a) used as an internal standard.	Female J0 - 1	Oncorhynchus mykiss	GSM38869 extract 1	total RNA	GSM38869 LE 1	1	GSM38869	assay	Rainbow trout sex differentiation cascade-related genes investigated using RT-PCR	P-GSE2151-1	GSM38869_sample_table.txt norm	GSM38869_sample_table.txt	ftp://ftp.ebi.ac.uk/pub/databases/microarray/data/experiment/GEOD/E-GEOD-2151/E-GEOD-2151.processed.1.zip
GSM38868 1	Large-scale temporal gene expression profiling during gonadal differentiation and early gametogenesis in rainbow-trout	Animals and samplings:	Research involving animal experimentation has been approved by the author's institution (authorization number 35-14) and conforms to NIH guidelines. All-male and all-female rainbow trout populations were obtained from the INRA experimental fish farm (Drennec, France) using breeders from the Mirwart strain. At 55 days post-fertilization (55 dpf, i.e., the onset of the free swimming), a male and female batch of 1500 fry each were transferred in 0.3 m3 tanks with recirculating water conditions. They were held at 12°C under constant photoperiod (12h light : 12h dark) and fed ad libitum with a commercial diet (dry pellet food, BiomarTM, Brande, Denmark). In each group, from 20 to 100 gonads, depending on the age of the fish, were sampled and pooled in duplicates at various stages of development : the onset of the free swimming period after complete yolk resumption (Day 0 = D0), D7, D12 (around the first occurrence of oocyte meiosis), D27 (first ovarian lamellar structures), D60 (first previtellogenic oocytes), D90 and D110. There were immediately frozen in liquid nitrogen and stored at -80°C until RNA extraction. Additional gonads were sampled at the same dates for histological analysis performed as previously described.	Total RNA extraction and Reverse Transcription:	Total RNA was extracted using TRIzol reagent (Invitrogen, Cergy Pontoise, France). Total RNA concentration was determined with an Agilent 2100 Bioanalyzer and the RNA 6000 LabChip kit (Agilent Technologies, Stockport, UK) according to the manufacturer's instructions. For cDNA synthesis, 1µg of RNA was denaturated in the presence of random hexamers (0.5µg) for 5 min at 70°C, and then chilled on ice. Reverse transcription (RT) was performed at 37°C for 1 h using M-MLV reverse transcriptase (Promega, Madison, WI) as described by the manufacturer.	Primers design:	Trout candidate gene homologues were searched in international public databanks using a reciprocal blast hit strategy. Candidate genes were chosen according to a bibliographic analysis showing their direct or indirect involvement in the sex differentiation cascade in vertebrates. All the primers were purchased from Eurogentec (Eurogentec, Seraing, Belgium). These primers were manually designed respecting whenever possible the following restriction parameters : 21-23 bp length, no more than 4 identical successive nucleotides, 30-70% GC content, a maximum of 2 G or C among the five 3'-end bases, no primer dimers and a short amplicon size (70-150 bp). Secondary structures were searched with DNA mfold (http://bioinfo.math.rpi.edu/~mfold/DNA/form1.cgi). Whenever possible, each pair was chosen with at least one primer flanking an intron-exon boundary, in order to prevent genomic amplification.	Real-time RT-PCR	Real-time RT-PCR was carried out on an iCycler iQTM (BioRad, Hercules, CA). Reactions were performed in 20µl with 300nM of each primer, 5µl of a 1/50 dilution of the RT reaction and the SYBER-Green PCR master Mix (Eurogentec) according to the manufacturer's instructions. After two incubation steps (50°C 10 min, 95°C 2 min), the thermal cycling protocol was 95°C for 10 min followed by 40 cycles of PCR (95°C 30 s, 60°C or 65°C 1 min). For each primer set the efficiency of the PCR reaction (linear equation : y = slope + intercept) was measured in triplicate on serial dilutions of the same cDNA sample (pool of reverse transcribed RNA samples). Real-time PCR efficiencies for each reaction were then calculated using the formula : Efficiency (E)=[10(1/slope)]-1. Melting curve analysis was also performed for each gene to check the specificity and identity of the RT-PCR products. The relative amount of the target RNA called the Starting Quantity (SQ) was then determined using the I-Cycler IQ software by comparison with the corresponding standard curve for each sample run in duplicate. SQ were calculated as follow : SQ = [10((Ct -intercept)/slope))]-1 where Ct is the Cycle threshold of the unknown sample. Each transcript level was then normalized by division with the expression values of the constitutive elongation factor 1 alpha (EF1a) used as an internal standard.	Male J110 - 2	Oncorhynchus mykiss	GSM38868 extract 1	total RNA	GSM38868 LE 1	1	GSM38868	assay	Rainbow trout sex differentiation cascade-related genes investigated using RT-PCR	P-GSE2151-1	GSM38868_sample_table.txt norm	GSM38868_sample_table.txt	ftp://ftp.ebi.ac.uk/pub/databases/microarray/data/experiment/GEOD/E-GEOD-2151/E-GEOD-2151.processed.1.zip
GSM38867 1	Large-scale temporal gene expression profiling during gonadal differentiation and early gametogenesis in rainbow-trout	Animals and samplings:	Research involving animal experimentation has been approved by the author's institution (authorization number 35-14) and conforms to NIH guidelines. All-male and all-female rainbow trout populations were obtained from the INRA experimental fish farm (Drennec, France) using breeders from the Mirwart strain. At 55 days post-fertilization (55 dpf, i.e., the onset of the free swimming), a male and female batch of 1500 fry each were transferred in 0.3 m3 tanks with recirculating water conditions. They were held at 12°C under constant photoperiod (12h light : 12h dark) and fed ad libitum with a commercial diet (dry pellet food, BiomarTM, Brande, Denmark). In each group, from 20 to 100 gonads, depending on the age of the fish, were sampled and pooled in duplicates at various stages of development : the onset of the free swimming period after complete yolk resumption (Day 0 = D0), D7, D12 (around the first occurrence of oocyte meiosis), D27 (first ovarian lamellar structures), D60 (first previtellogenic oocytes), D90 and D110. There were immediately frozen in liquid nitrogen and stored at -80°C until RNA extraction. Additional gonads were sampled at the same dates for histological analysis performed as previously described.	Total RNA extraction and Reverse Transcription:	Total RNA was extracted using TRIzol reagent (Invitrogen, Cergy Pontoise, France). Total RNA concentration was determined with an Agilent 2100 Bioanalyzer and the RNA 6000 LabChip kit (Agilent Technologies, Stockport, UK) according to the manufacturer's instructions. For cDNA synthesis, 1µg of RNA was denaturated in the presence of random hexamers (0.5µg) for 5 min at 70°C, and then chilled on ice. Reverse transcription (RT) was performed at 37°C for 1 h using M-MLV reverse transcriptase (Promega, Madison, WI) as described by the manufacturer.	Primers design:	Trout candidate gene homologues were searched in international public databanks using a reciprocal blast hit strategy. Candidate genes were chosen according to a bibliographic analysis showing their direct or indirect involvement in the sex differentiation cascade in vertebrates. All the primers were purchased from Eurogentec (Eurogentec, Seraing, Belgium). These primers were manually designed respecting whenever possible the following restriction parameters : 21-23 bp length, no more than 4 identical successive nucleotides, 30-70% GC content, a maximum of 2 G or C among the five 3'-end bases, no primer dimers and a short amplicon size (70-150 bp). Secondary structures were searched with DNA mfold (http://bioinfo.math.rpi.edu/~mfold/DNA/form1.cgi). Whenever possible, each pair was chosen with at least one primer flanking an intron-exon boundary, in order to prevent genomic amplification.	Real-time RT-PCR	Real-time RT-PCR was carried out on an iCycler iQTM (BioRad, Hercules, CA). Reactions were performed in 20µl with 300nM of each primer, 5µl of a 1/50 dilution of the RT reaction and the SYBER-Green PCR master Mix (Eurogentec) according to the manufacturer's instructions. After two incubation steps (50°C 10 min, 95°C 2 min), the thermal cycling protocol was 95°C for 10 min followed by 40 cycles of PCR (95°C 30 s, 60°C or 65°C 1 min). For each primer set the efficiency of the PCR reaction (linear equation : y = slope + intercept) was measured in triplicate on serial dilutions of the same cDNA sample (pool of reverse transcribed RNA samples). Real-time PCR efficiencies for each reaction were then calculated using the formula : Efficiency (E)=[10(1/slope)]-1. Melting curve analysis was also performed for each gene to check the specificity and identity of the RT-PCR products. The relative amount of the target RNA called the Starting Quantity (SQ) was then determined using the I-Cycler IQ software by comparison with the corresponding standard curve for each sample run in duplicate. SQ were calculated as follow : SQ = [10((Ct -intercept)/slope))]-1 where Ct is the Cycle threshold of the unknown sample. Each transcript level was then normalized by division with the expression values of the constitutive elongation factor 1 alpha (EF1a) used as an internal standard.	Male J110 - 1	Oncorhynchus mykiss	GSM38867 extract 1	total RNA	GSM38867 LE 1	1	GSM38867	assay	Rainbow trout sex differentiation cascade-related genes investigated using RT-PCR	P-GSE2151-1	GSM38867_sample_table.txt norm	GSM38867_sample_table.txt	ftp://ftp.ebi.ac.uk/pub/databases/microarray/data/experiment/GEOD/E-GEOD-2151/E-GEOD-2151.processed.1.zip
GSM38866 1	Large-scale temporal gene expression profiling during gonadal differentiation and early gametogenesis in rainbow-trout	Animals and samplings:	Research involving animal experimentation has been approved by the author's institution (authorization number 35-14) and conforms to NIH guidelines. All-male and all-female rainbow trout populations were obtained from the INRA experimental fish farm (Drennec, France) using breeders from the Mirwart strain. At 55 days post-fertilization (55 dpf, i.e., the onset of the free swimming), a male and female batch of 1500 fry each were transferred in 0.3 m3 tanks with recirculating water conditions. They were held at 12°C under constant photoperiod (12h light : 12h dark) and fed ad libitum with a commercial diet (dry pellet food, BiomarTM, Brande, Denmark). In each group, from 20 to 100 gonads, depending on the age of the fish, were sampled and pooled in duplicates at various stages of development : the onset of the free swimming period after complete yolk resumption (Day 0 = D0), D7, D12 (around the first occurrence of oocyte meiosis), D27 (first ovarian lamellar structures), D60 (first previtellogenic oocytes), D90 and D110. There were immediately frozen in liquid nitrogen and stored at -80°C until RNA extraction. Additional gonads were sampled at the same dates for histological analysis performed as previously described.	Total RNA extraction and Reverse Transcription:	Total RNA was extracted using TRIzol reagent (Invitrogen, Cergy Pontoise, France). Total RNA concentration was determined with an Agilent 2100 Bioanalyzer and the RNA 6000 LabChip kit (Agilent Technologies, Stockport, UK) according to the manufacturer's instructions. For cDNA synthesis, 1µg of RNA was denaturated in the presence of random hexamers (0.5µg) for 5 min at 70°C, and then chilled on ice. Reverse transcription (RT) was performed at 37°C for 1 h using M-MLV reverse transcriptase (Promega, Madison, WI) as described by the manufacturer.	Primers design:	Trout candidate gene homologues were searched in international public databanks using a reciprocal blast hit strategy. Candidate genes were chosen according to a bibliographic analysis showing their direct or indirect involvement in the sex differentiation cascade in vertebrates. All the primers were purchased from Eurogentec (Eurogentec, Seraing, Belgium). These primers were manually designed respecting whenever possible the following restriction parameters : 21-23 bp length, no more than 4 identical successive nucleotides, 30-70% GC content, a maximum of 2 G or C among the five 3'-end bases, no primer dimers and a short amplicon size (70-150 bp). Secondary structures were searched with DNA mfold (http://bioinfo.math.rpi.edu/~mfold/DNA/form1.cgi). Whenever possible, each pair was chosen with at least one primer flanking an intron-exon boundary, in order to prevent genomic amplification.	Real-time RT-PCR	Real-time RT-PCR was carried out on an iCycler iQTM (BioRad, Hercules, CA). Reactions were performed in 20µl with 300nM of each primer, 5µl of a 1/50 dilution of the RT reaction and the SYBER-Green PCR master Mix (Eurogentec) according to the manufacturer's instructions. After two incubation steps (50°C 10 min, 95°C 2 min), the thermal cycling protocol was 95°C for 10 min followed by 40 cycles of PCR (95°C 30 s, 60°C or 65°C 1 min). For each primer set the efficiency of the PCR reaction (linear equation : y = slope + intercept) was measured in triplicate on serial dilutions of the same cDNA sample (pool of reverse transcribed RNA samples). Real-time PCR efficiencies for each reaction were then calculated using the formula : Efficiency (E)=[10(1/slope)]-1. Melting curve analysis was also performed for each gene to check the specificity and identity of the RT-PCR products. The relative amount of the target RNA called the Starting Quantity (SQ) was then determined using the I-Cycler IQ software by comparison with the corresponding standard curve for each sample run in duplicate. SQ were calculated as follow : SQ = [10((Ct -intercept)/slope))]-1 where Ct is the Cycle threshold of the unknown sample. Each transcript level was then normalized by division with the expression values of the constitutive elongation factor 1 alpha (EF1a) used as an internal standard.	Male J90 - 2	Oncorhynchus mykiss	GSM38866 extract 1	total RNA	GSM38866 LE 1	1	GSM38866	assay	Rainbow trout sex differentiation cascade-related genes investigated using RT-PCR	P-GSE2151-1	GSM38866_sample_table.txt norm	GSM38866_sample_table.txt	ftp://ftp.ebi.ac.uk/pub/databases/microarray/data/experiment/GEOD/E-GEOD-2151/E-GEOD-2151.processed.1.zip
GSM38865 1	Large-scale temporal gene expression profiling during gonadal differentiation and early gametogenesis in rainbow-trout	Animals and samplings:	Research involving animal experimentation has been approved by the author's institution (authorization number 35-14) and conforms to NIH guidelines. All-male and all-female rainbow trout populations were obtained from the INRA experimental fish farm (Drennec, France) using breeders from the Mirwart strain. At 55 days post-fertilization (55 dpf, i.e., the onset of the free swimming), a male and female batch of 1500 fry each were transferred in 0.3 m3 tanks with recirculating water conditions. They were held at 12°C under constant photoperiod (12h light : 12h dark) and fed ad libitum with a commercial diet (dry pellet food, BiomarTM, Brande, Denmark). In each group, from 20 to 100 gonads, depending on the age of the fish, were sampled and pooled in duplicates at various stages of development : the onset of the free swimming period after complete yolk resumption (Day 0 = D0), D7, D12 (around the first occurrence of oocyte meiosis), D27 (first ovarian lamellar structures), D60 (first previtellogenic oocytes), D90 and D110. There were immediately frozen in liquid nitrogen and stored at -80°C until RNA extraction. Additional gonads were sampled at the same dates for histological analysis performed as previously described.	Total RNA extraction and Reverse Transcription:	Total RNA was extracted using TRIzol reagent (Invitrogen, Cergy Pontoise, France). Total RNA concentration was determined with an Agilent 2100 Bioanalyzer and the RNA 6000 LabChip kit (Agilent Technologies, Stockport, UK) according to the manufacturer's instructions. For cDNA synthesis, 1µg of RNA was denaturated in the presence of random hexamers (0.5µg) for 5 min at 70°C, and then chilled on ice. Reverse transcription (RT) was performed at 37°C for 1 h using M-MLV reverse transcriptase (Promega, Madison, WI) as described by the manufacturer.	Primers design:	Trout candidate gene homologues were searched in international public databanks using a reciprocal blast hit strategy. Candidate genes were chosen according to a bibliographic analysis showing their direct or indirect involvement in the sex differentiation cascade in vertebrates. All the primers were purchased from Eurogentec (Eurogentec, Seraing, Belgium). These primers were manually designed respecting whenever possible the following restriction parameters : 21-23 bp length, no more than 4 identical successive nucleotides, 30-70% GC content, a maximum of 2 G or C among the five 3'-end bases, no primer dimers and a short amplicon size (70-150 bp). Secondary structures were searched with DNA mfold (http://bioinfo.math.rpi.edu/~mfold/DNA/form1.cgi). Whenever possible, each pair was chosen with at least one primer flanking an intron-exon boundary, in order to prevent genomic amplification.	Real-time RT-PCR	Real-time RT-PCR was carried out on an iCycler iQTM (BioRad, Hercules, CA). Reactions were performed in 20µl with 300nM of each primer, 5µl of a 1/50 dilution of the RT reaction and the SYBER-Green PCR master Mix (Eurogentec) according to the manufacturer's instructions. After two incubation steps (50°C 10 min, 95°C 2 min), the thermal cycling protocol was 95°C for 10 min followed by 40 cycles of PCR (95°C 30 s, 60°C or 65°C 1 min). For each primer set the efficiency of the PCR reaction (linear equation : y = slope + intercept) was measured in triplicate on serial dilutions of the same cDNA sample (pool of reverse transcribed RNA samples). Real-time PCR efficiencies for each reaction were then calculated using the formula : Efficiency (E)=[10(1/slope)]-1. Melting curve analysis was also performed for each gene to check the specificity and identity of the RT-PCR products. The relative amount of the target RNA called the Starting Quantity (SQ) was then determined using the I-Cycler IQ software by comparison with the corresponding standard curve for each sample run in duplicate. SQ were calculated as follow : SQ = [10((Ct -intercept)/slope))]-1 where Ct is the Cycle threshold of the unknown sample. Each transcript level was then normalized by division with the expression values of the constitutive elongation factor 1 alpha (EF1a) used as an internal standard.	Male J90 - 1	Oncorhynchus mykiss	GSM38865 extract 1	total RNA	GSM38865 LE 1	1	GSM38865	assay	Rainbow trout sex differentiation cascade-related genes investigated using RT-PCR	P-GSE2151-1	GSM38865_sample_table.txt norm	GSM38865_sample_table.txt	ftp://ftp.ebi.ac.uk/pub/databases/microarray/data/experiment/GEOD/E-GEOD-2151/E-GEOD-2151.processed.1.zip
GSM38864 1	Large-scale temporal gene expression profiling during gonadal differentiation and early gametogenesis in rainbow-trout	Animals and samplings:	Research involving animal experimentation has been approved by the author's institution (authorization number 35-14) and conforms to NIH guidelines. All-male and all-female rainbow trout populations were obtained from the INRA experimental fish farm (Drennec, France) using breeders from the Mirwart strain. At 55 days post-fertilization (55 dpf, i.e., the onset of the free swimming), a male and female batch of 1500 fry each were transferred in 0.3 m3 tanks with recirculating water conditions. They were held at 12°C under constant photoperiod (12h light : 12h dark) and fed ad libitum with a commercial diet (dry pellet food, BiomarTM, Brande, Denmark). In each group, from 20 to 100 gonads, depending on the age of the fish, were sampled and pooled in duplicates at various stages of development : the onset of the free swimming period after complete yolk resumption (Day 0 = D0), D7, D12 (around the first occurrence of oocyte meiosis), D27 (first ovarian lamellar structures), D60 (first previtellogenic oocytes), D90 and D110. There were immediately frozen in liquid nitrogen and stored at -80°C until RNA extraction. Additional gonads were sampled at the same dates for histological analysis performed as previously described.	Total RNA extraction and Reverse Transcription:	Total RNA was extracted using TRIzol reagent (Invitrogen, Cergy Pontoise, France). Total RNA concentration was determined with an Agilent 2100 Bioanalyzer and the RNA 6000 LabChip kit (Agilent Technologies, Stockport, UK) according to the manufacturer's instructions. For cDNA synthesis, 1µg of RNA was denaturated in the presence of random hexamers (0.5µg) for 5 min at 70°C, and then chilled on ice. Reverse transcription (RT) was performed at 37°C for 1 h using M-MLV reverse transcriptase (Promega, Madison, WI) as described by the manufacturer.	Primers design:	Trout candidate gene homologues were searched in international public databanks using a reciprocal blast hit strategy. Candidate genes were chosen according to a bibliographic analysis showing their direct or indirect involvement in the sex differentiation cascade in vertebrates. All the primers were purchased from Eurogentec (Eurogentec, Seraing, Belgium). These primers were manually designed respecting whenever possible the following restriction parameters : 21-23 bp length, no more than 4 identical successive nucleotides, 30-70% GC content, a maximum of 2 G or C among the five 3'-end bases, no primer dimers and a short amplicon size (70-150 bp). Secondary structures were searched with DNA mfold (http://bioinfo.math.rpi.edu/~mfold/DNA/form1.cgi). Whenever possible, each pair was chosen with at least one primer flanking an intron-exon boundary, in order to prevent genomic amplification.	Real-time RT-PCR	Real-time RT-PCR was carried out on an iCycler iQTM (BioRad, Hercules, CA). Reactions were performed in 20µl with 300nM of each primer, 5µl of a 1/50 dilution of the RT reaction and the SYBER-Green PCR master Mix (Eurogentec) according to the manufacturer's instructions. After two incubation steps (50°C 10 min, 95°C 2 min), the thermal cycling protocol was 95°C for 10 min followed by 40 cycles of PCR (95°C 30 s, 60°C or 65°C 1 min). For each primer set the efficiency of the PCR reaction (linear equation : y = slope + intercept) was measured in triplicate on serial dilutions of the same cDNA sample (pool of reverse transcribed RNA samples). Real-time PCR efficiencies for each reaction were then calculated using the formula : Efficiency (E)=[10(1/slope)]-1. Melting curve analysis was also performed for each gene to check the specificity and identity of the RT-PCR products. The relative amount of the target RNA called the Starting Quantity (SQ) was then determined using the I-Cycler IQ software by comparison with the corresponding standard curve for each sample run in duplicate. SQ were calculated as follow : SQ = [10((Ct -intercept)/slope))]-1 where Ct is the Cycle threshold of the unknown sample. Each transcript level was then normalized by division with the expression values of the constitutive elongation factor 1 alpha (EF1a) used as an internal standard.	Male J60 - 2	Oncorhynchus mykiss	GSM38864 extract 1	total RNA	GSM38864 LE 1	1	GSM38864	assay	Rainbow trout sex differentiation cascade-related genes investigated using RT-PCR	P-GSE2151-1	GSM38864_sample_table.txt norm	GSM38864_sample_table.txt	ftp://ftp.ebi.ac.uk/pub/databases/microarray/data/experiment/GEOD/E-GEOD-2151/E-GEOD-2151.processed.1.zip
GSM38863 1	Large-scale temporal gene expression profiling during gonadal differentiation and early gametogenesis in rainbow-trout	Animals and samplings:	Research involving animal experimentation has been approved by the author's institution (authorization number 35-14) and conforms to NIH guidelines. All-male and all-female rainbow trout populations were obtained from the INRA experimental fish farm (Drennec, France) using breeders from the Mirwart strain. At 55 days post-fertilization (55 dpf, i.e., the onset of the free swimming), a male and female batch of 1500 fry each were transferred in 0.3 m3 tanks with recirculating water conditions. They were held at 12°C under constant photoperiod (12h light : 12h dark) and fed ad libitum with a commercial diet (dry pellet food, BiomarTM, Brande, Denmark). In each group, from 20 to 100 gonads, depending on the age of the fish, were sampled and pooled in duplicates at various stages of development : the onset of the free swimming period after complete yolk resumption (Day 0 = D0), D7, D12 (around the first occurrence of oocyte meiosis), D27 (first ovarian lamellar structures), D60 (first previtellogenic oocytes), D90 and D110. There were immediately frozen in liquid nitrogen and stored at -80°C until RNA extraction. Additional gonads were sampled at the same dates for histological analysis performed as previously described.	Total RNA extraction and Reverse Transcription:	Total RNA was extracted using TRIzol reagent (Invitrogen, Cergy Pontoise, France). Total RNA concentration was determined with an Agilent 2100 Bioanalyzer and the RNA 6000 LabChip kit (Agilent Technologies, Stockport, UK) according to the manufacturer's instructions. For cDNA synthesis, 1µg of RNA was denaturated in the presence of random hexamers (0.5µg) for 5 min at 70°C, and then chilled on ice. Reverse transcription (RT) was performed at 37°C for 1 h using M-MLV reverse transcriptase (Promega, Madison, WI) as described by the manufacturer.	Primers design:	Trout candidate gene homologues were searched in international public databanks using a reciprocal blast hit strategy. Candidate genes were chosen according to a bibliographic analysis showing their direct or indirect involvement in the sex differentiation cascade in vertebrates. All the primers were purchased from Eurogentec (Eurogentec, Seraing, Belgium). These primers were manually designed respecting whenever possible the following restriction parameters : 21-23 bp length, no more than 4 identical successive nucleotides, 30-70% GC content, a maximum of 2 G or C among the five 3'-end bases, no primer dimers and a short amplicon size (70-150 bp). Secondary structures were searched with DNA mfold (http://bioinfo.math.rpi.edu/~mfold/DNA/form1.cgi). Whenever possible, each pair was chosen with at least one primer flanking an intron-exon boundary, in order to prevent genomic amplification.	Real-time RT-PCR	Real-time RT-PCR was carried out on an iCycler iQTM (BioRad, Hercules, CA). Reactions were performed in 20µl with 300nM of each primer, 5µl of a 1/50 dilution of the RT reaction and the SYBER-Green PCR master Mix (Eurogentec) according to the manufacturer's instructions. After two incubation steps (50°C 10 min, 95°C 2 min), the thermal cycling protocol was 95°C for 10 min followed by 40 cycles of PCR (95°C 30 s, 60°C or 65°C 1 min). For each primer set the efficiency of the PCR reaction (linear equation : y = slope + intercept) was measured in triplicate on serial dilutions of the same cDNA sample (pool of reverse transcribed RNA samples). Real-time PCR efficiencies for each reaction were then calculated using the formula : Efficiency (E)=[10(1/slope)]-1. Melting curve analysis was also performed for each gene to check the specificity and identity of the RT-PCR products. The relative amount of the target RNA called the Starting Quantity (SQ) was then determined using the I-Cycler IQ software by comparison with the corresponding standard curve for each sample run in duplicate. SQ were calculated as follow : SQ = [10((Ct -intercept)/slope))]-1 where Ct is the Cycle threshold of the unknown sample. Each transcript level was then normalized by division with the expression values of the constitutive elongation factor 1 alpha (EF1a) used as an internal standard.	Male J60 - 1	Oncorhynchus mykiss	GSM38863 extract 1	total RNA	GSM38863 LE 1	1	GSM38863	assay	Rainbow trout sex differentiation cascade-related genes investigated using RT-PCR	P-GSE2151-1	GSM38863_sample_table.txt norm	GSM38863_sample_table.txt	ftp://ftp.ebi.ac.uk/pub/databases/microarray/data/experiment/GEOD/E-GEOD-2151/E-GEOD-2151.processed.1.zip
GSM38862 1	Large-scale temporal gene expression profiling during gonadal differentiation and early gametogenesis in rainbow-trout	Animals and samplings:	Research involving animal experimentation has been approved by the author's institution (authorization number 35-14) and conforms to NIH guidelines. All-male and all-female rainbow trout populations were obtained from the INRA experimental fish farm (Drennec, France) using breeders from the Mirwart strain. At 55 days post-fertilization (55 dpf, i.e., the onset of the free swimming), a male and female batch of 1500 fry each were transferred in 0.3 m3 tanks with recirculating water conditions. They were held at 12°C under constant photoperiod (12h light : 12h dark) and fed ad libitum with a commercial diet (dry pellet food, BiomarTM, Brande, Denmark). In each group, from 20 to 100 gonads, depending on the age of the fish, were sampled and pooled in duplicates at various stages of development : the onset of the free swimming period after complete yolk resumption (Day 0 = D0), D7, D12 (around the first occurrence of oocyte meiosis), D27 (first ovarian lamellar structures), D60 (first previtellogenic oocytes), D90 and D110. There were immediately frozen in liquid nitrogen and stored at -80°C until RNA extraction. Additional gonads were sampled at the same dates for histological analysis performed as previously described.	Total RNA extraction and Reverse Transcription:	Total RNA was extracted using TRIzol reagent (Invitrogen, Cergy Pontoise, France). Total RNA concentration was determined with an Agilent 2100 Bioanalyzer and the RNA 6000 LabChip kit (Agilent Technologies, Stockport, UK) according to the manufacturer's instructions. For cDNA synthesis, 1µg of RNA was denaturated in the presence of random hexamers (0.5µg) for 5 min at 70°C, and then chilled on ice. Reverse transcription (RT) was performed at 37°C for 1 h using M-MLV reverse transcriptase (Promega, Madison, WI) as described by the manufacturer.	Primers design:	Trout candidate gene homologues were searched in international public databanks using a reciprocal blast hit strategy. Candidate genes were chosen according to a bibliographic analysis showing their direct or indirect involvement in the sex differentiation cascade in vertebrates. All the primers were purchased from Eurogentec (Eurogentec, Seraing, Belgium). These primers were manually designed respecting whenever possible the following restriction parameters : 21-23 bp length, no more than 4 identical successive nucleotides, 30-70% GC content, a maximum of 2 G or C among the five 3'-end bases, no primer dimers and a short amplicon size (70-150 bp). Secondary structures were searched with DNA mfold (http://bioinfo.math.rpi.edu/~mfold/DNA/form1.cgi). Whenever possible, each pair was chosen with at least one primer flanking an intron-exon boundary, in order to prevent genomic amplification.	Real-time RT-PCR	Real-time RT-PCR was carried out on an iCycler iQTM (BioRad, Hercules, CA). Reactions were performed in 20µl with 300nM of each primer, 5µl of a 1/50 dilution of the RT reaction and the SYBER-Green PCR master Mix (Eurogentec) according to the manufacturer's instructions. After two incubation steps (50°C 10 min, 95°C 2 min), the thermal cycling protocol was 95°C for 10 min followed by 40 cycles of PCR (95°C 30 s, 60°C or 65°C 1 min). For each primer set the efficiency of the PCR reaction (linear equation : y = slope + intercept) was measured in triplicate on serial dilutions of the same cDNA sample (pool of reverse transcribed RNA samples). Real-time PCR efficiencies for each reaction were then calculated using the formula : Efficiency (E)=[10(1/slope)]-1. Melting curve analysis was also performed for each gene to check the specificity and identity of the RT-PCR products. The relative amount of the target RNA called the Starting Quantity (SQ) was then determined using the I-Cycler IQ software by comparison with the corresponding standard curve for each sample run in duplicate. SQ were calculated as follow : SQ = [10((Ct -intercept)/slope))]-1 where Ct is the Cycle threshold of the unknown sample. Each transcript level was then normalized by division with the expression values of the constitutive elongation factor 1 alpha (EF1a) used as an internal standard.	Male J27 - 2	Oncorhynchus mykiss	GSM38862 extract 1	total RNA	GSM38862 LE 1	1	GSM38862	assay	Rainbow trout sex differentiation cascade-related genes investigated using RT-PCR	P-GSE2151-1	GSM38862_sample_table.txt norm	GSM38862_sample_table.txt	ftp://ftp.ebi.ac.uk/pub/databases/microarray/data/experiment/GEOD/E-GEOD-2151/E-GEOD-2151.processed.1.zip
GSM38861 1	Large-scale temporal gene expression profiling during gonadal differentiation and early gametogenesis in rainbow-trout	Animals and samplings:	Research involving animal experimentation has been approved by the author's institution (authorization number 35-14) and conforms to NIH guidelines. All-male and all-female rainbow trout populations were obtained from the INRA experimental fish farm (Drennec, France) using breeders from the Mirwart strain. At 55 days post-fertilization (55 dpf, i.e., the onset of the free swimming), a male and female batch of 1500 fry each were transferred in 0.3 m3 tanks with recirculating water conditions. They were held at 12°C under constant photoperiod (12h light : 12h dark) and fed ad libitum with a commercial diet (dry pellet food, BiomarTM, Brande, Denmark). In each group, from 20 to 100 gonads, depending on the age of the fish, were sampled and pooled in duplicates at various stages of development : the onset of the free swimming period after complete yolk resumption (Day 0 = D0), D7, D12 (around the first occurrence of oocyte meiosis), D27 (first ovarian lamellar structures), D60 (first previtellogenic oocytes), D90 and D110. There were immediately frozen in liquid nitrogen and stored at -80°C until RNA extraction. Additional gonads were sampled at the same dates for histological analysis performed as previously described.	Total RNA extraction and Reverse Transcription:	Total RNA was extracted using TRIzol reagent (Invitrogen, Cergy Pontoise, France). Total RNA concentration was determined with an Agilent 2100 Bioanalyzer and the RNA 6000 LabChip kit (Agilent Technologies, Stockport, UK) according to the manufacturer's instructions. For cDNA synthesis, 1µg of RNA was denaturated in the presence of random hexamers (0.5µg) for 5 min at 70°C, and then chilled on ice. Reverse transcription (RT) was performed at 37°C for 1 h using M-MLV reverse transcriptase (Promega, Madison, WI) as described by the manufacturer.	Primers design:	Trout candidate gene homologues were searched in international public databanks using a reciprocal blast hit strategy. Candidate genes were chosen according to a bibliographic analysis showing their direct or indirect involvement in the sex differentiation cascade in vertebrates. All the primers were purchased from Eurogentec (Eurogentec, Seraing, Belgium). These primers were manually designed respecting whenever possible the following restriction parameters : 21-23 bp length, no more than 4 identical successive nucleotides, 30-70% GC content, a maximum of 2 G or C among the five 3'-end bases, no primer dimers and a short amplicon size (70-150 bp). Secondary structures were searched with DNA mfold (http://bioinfo.math.rpi.edu/~mfold/DNA/form1.cgi). Whenever possible, each pair was chosen with at least one primer flanking an intron-exon boundary, in order to prevent genomic amplification.	Real-time RT-PCR	Real-time RT-PCR was carried out on an iCycler iQTM (BioRad, Hercules, CA). Reactions were performed in 20µl with 300nM of each primer, 5µl of a 1/50 dilution of the RT reaction and the SYBER-Green PCR master Mix (Eurogentec) according to the manufacturer's instructions. After two incubation steps (50°C 10 min, 95°C 2 min), the thermal cycling protocol was 95°C for 10 min followed by 40 cycles of PCR (95°C 30 s, 60°C or 65°C 1 min). For each primer set the efficiency of the PCR reaction (linear equation : y = slope + intercept) was measured in triplicate on serial dilutions of the same cDNA sample (pool of reverse transcribed RNA samples). Real-time PCR efficiencies for each reaction were then calculated using the formula : Efficiency (E)=[10(1/slope)]-1. Melting curve analysis was also performed for each gene to check the specificity and identity of the RT-PCR products. The relative amount of the target RNA called the Starting Quantity (SQ) was then determined using the I-Cycler IQ software by comparison with the corresponding standard curve for each sample run in duplicate. SQ were calculated as follow : SQ = [10((Ct -intercept)/slope))]-1 where Ct is the Cycle threshold of the unknown sample. Each transcript level was then normalized by division with the expression values of the constitutive elongation factor 1 alpha (EF1a) used as an internal standard.	Male J0 - 1	Oncorhynchus mykiss	GSM38861 extract 1	total RNA	GSM38861 LE 1	1	GSM38861	assay	Rainbow trout sex differentiation cascade-related genes investigated using RT-PCR	P-GSE2151-1	GSM38861_sample_table.txt norm	GSM38861_sample_table.txt	ftp://ftp.ebi.ac.uk/pub/databases/microarray/data/experiment/GEOD/E-GEOD-2151/E-GEOD-2151.processed.1.zip