Comment[ArrayExpressAccession] E-GEOD-41908 MAGE-TAB Version 1.1 Public Release Date 2012-10-31 Investigation Title Transcriptome profiling of wild type primordial germ cells (PGC) by RNAseq Comment[Submitted Name] Transcriptome profiling of wild type primordial germ cells (PGC) by RNAseq Experiment Description Purpose: To identify molecular pathways underlying epigenetic reprogramming in early germ cell precursors, we examined global gene expression of wild type primordial germ cells using mRNA sequencing. Methods: Given the limited number of PGCs collected from E9.5 to E13.5 (ranging from 300 to 5000), we used a low-input RNA sequencing method, Smart-Seq. RNA libraries were pooled and sequenced by Illumina Hiseq. Results: We generated >22 million uniquely mapped reads per sample and identified >10 thousand transcripts per genotype (RPKM>0.1). Hierarchical clustering and correlation analysis on gene expression indicates samples were clearly separated according to their genotypes with Spearman correlation coefficient of 0.98/0.99 within biological replicates. Compared with E9.5 PGCs, 479 genes were significantly up-regulated and 248 genes were down-regulated in E11.5 PGCs. When compared with E11.5 PGCs, male E13.5 PGCs had 362 up-regulated, and 239 down-regulated genes, whereas female E13.5 PGCs had 1163 up-regulated and 333 down-regulated genes. Overall, the number of up-regulated genes was greater than that of the down-regulated genes in every comparison, suggesting that gene expression is generally activated during PGC reprogramming. mRNA profiles of primordial germ cells derived from developmental embryos stages (E9.5, E11.5 and E13.5) were generated by deep sequencing, in duplicates (E9.5 and E11.5) or triplicates (E13.5f and E13.5m), using Illumina Hiseq. Term Source Name ArrayExpress EFO Term Source File http://www.ebi.ac.uk/arrayexpress/ http://www.ebi.ac.uk/efo/efo.owl Person Last Name Diep Person First Name Dinh Person Mid Initials Hue Person Email hdinhdp@gmail.com Person Affiliation University of California, San Diego Person Address Bioengineering, University of California, San Diego, 9500 Gilman Dr Mailcode: 0412, La Jolla, CA, USA Person Roles submitter Protocol Name P-GSE41908-1 P-GSE41908-3 P-GSE41908-2 P-GSE41908-4 Protocol Description Total RNA was purified from 500-2,000 PGCs using ZR RNA microprep kit (Zymo Research, UAS). The cDNA synthesis and amplification was performed with the SMARTer ultra low input RNA kit (Clontech, US). The amplified cDNA (2-70 ng) was then fragmented by Covaris S2 sonicator (Covaris, US) and converted to sequencing libraries following the Illumina’s construction protocol for low input DNA (Illumina, US). Barcoded libraries were pooled and sequenced in three lanes of Illumina Hiseq 2000 instrument. Total RNA was purified from 400-5,000 PGCs using ZR RNA microprep kit (Zymo Research, UAS). The cDNA synthesis and amplification was performed with the SMARTer ultra low input RNA kit (Clontech, US). The amplified cDNA (2-70 ng) was then fragmented by Covaris S2 sonicator (Covaris, US) and converted to sequencing libraries following the Illumina’s construction protocol for low input DNA (Illumina, US). Barcoded libraries were pooled and sequenced in three lanes of Illumina Hiseq 2000 instrument. mRNA-seq reads generated from each sample were aligned to the mouse genome (mm9, NCBI build 37) with Bowtie/Tophat v1.3.1 (http://tophat.cbcb.umd.edu) which allows mapping across splice sites by reads segmentation. All programs were used with default setting unless otherwise specified. Mapped reads (83-84% of total reads) were subsequently assembled into transcripts guided by reference annotation (mm9, USCS gene annotation) with Cufflinks v1.0.3 (http://cufflinks.cbcb.umd.edu). Expression level of each transcript was quantified with normalized FPKM (fragments per kilobase of exon per million mapped fragments). Two or three biological replicates were used in each genotype to identify transcripts that showed significant differences at a FDR cutoff <0.05 between wild type and Tet1Gt/Gt by Cuffdiff v1.0.3. Functional annotation of significantly different transcripts and enrichment analysis was performed with DAVID (http://david.abcc.ncifcrf.gov). Genome_build: mm9 Supplementary_files_format_and_content: Text file include average FPKM for wildtype and average FPKM for Tet1-knockout, and statistics generated by CuffLinks (cuffdiff) mRNA-seq reads generated from each sample were aligned to the mouse genome (mm9, NCBI build 37) with Bowtie/Tophat v1.3.1 (http://tophat.cbcb.umd.edu) which allows mapping across splice sites by reads segmentation. All programs were used with default setting unless otherwise specified. Mapped reads (94-95% of total reads) were subsequently assembled into transcripts guided by reference annotation (mm9, USCS gene annotation) with Cufflinks v1.2.1 (http://cufflinks.cbcb.umd.edu). Expression level of each transcript was quantified with normalized FPKM (fragments per kilobase of exon per million mapped fragments). Two or three biological replicates were used in each genotype to identify transcripts that showed significant differences at a FDR cutoff <0.05 between wild type and Tet1Gt/Gt by Cuffdiff v1.2.1. Functional annotation of significantly different transcripts and enrichment analysis was performed with DAVID (http://david.abcc.ncifcrf.gov). Genome_build: mm9 Supplementary_files_format_and_content: tab-delimited text files include average RPKM for different developmental stages and statistics generated by CuffLinks (cuffdiff) Protocol Type nucleic acid library construction protocol nucleic acid library construction protocol feature_extraction feature_extraction Experimental Factor Name GENOTYPE AGE Experimental Factor Type genotype age Publication Title Tet1 controls meiosis by regulating meiotic gene expression. Publication Author List Yamaguchi S, Hong K, Liu R, Shen L, Inoue A, Diep D, Zhang K, Zhang Y PubMed ID 23151479 Publication DOI 10.1038/nature11709 Comment[SecondaryAccession] GSE41908 Comment[GEOReleaseDate] 2012-10-31 Comment[ArrayExpressSubmissionDate] 2012-10-29 Comment[GEOLastUpdateDate] 2013-03-01 Comment[AEExperimentType] RNA-seq of coding RNA Comment[AdditionalFile:Data1] GSE41908_E11.5_E13.5f_cuffdiff1.2.txt Comment[AdditionalFile:Data2] GSE41908_E11.5_E13.5m_cuffdiff1.2.txt Comment[AdditionalFile:Data3] GSE41908_E9.5_E11.5_cuffdiff1.2.txt Comment[AdditionalFile:Data4] GSE41908_RNAseq_cuffdiff.txt Comment[SecondaryAccession] SRP016882 Comment[SequenceDataURI] http://www.ebi.ac.uk/ena/data/view/SRR609722-SRR609736,SRR648686-SRR648697,SRR649335-SRR649343 SDRF File E-GEOD-41908.sdrf.txt