12 protocols
AccessionNameType
P-GSE42847-1
bioassay_data_transformation
ID_REF = VALUE = normalized log10 ratio Cy5/Cy3
P-GSE42847-8
feature_extraction
Microarray data was processed from raw data image files with Feature Extraction Software 9.5.3.1 (Agilent Technologies). The XDR function was used to extend the dynamic range. Processed data were subsequently imported into Rosetta Resolver 7.0 (Rosetta Biosoftware, Seattle, Washington) and subjected to default ratio error modelling. The Agilent Feature Extraction files contain the final log ratio data. The algorithms that were used to normalize the feature signal and calculate the log ratios are described in the Reference Guide for Agilent Feature Extraction Software v9.5. The standard settings as described for the GE2-v5_95 protocol in the Reference Guide were used. In order to calculate the normalized signal, the Rank Consistency Probes method was used and the Linear&LOWESSDyeNormFactor (= DyeNormalSignal/(BGSubSignal x LinearDyeNormFactor) was calculated as described on page 231 of the Reference Guide. The dye normalized signal was calculated as follows (page 232): DyeNormSignal = BGSubSignal x DyeNormFactor. The log10 ratio was subsequently calculated as follows (page 232): LogRatio = Log(rProcessedSignal/gProcessedSignal), where rProcessedSignal and gProcessedSignal are signals post dye normalization and post surrogate processing in the red and green channels, respectively. The surrogate values are calculated and used as the lowest limit of detection to replace the dye normalized signal when either the mean feature signal is less than the background signal or not significant when compared to the background signal, or when the mean signal is less than its background standard deviation (page 226).
P-GSE42847-7
image_aquisition
The arrays were scanned with DNA Microarray Scanner G2565CA from Agilent Technologies. The arrays were scanned twice with 10% PMT and 100% PMT laser power.
P-GSE42847-6
hybridization
The dual colour hybridization of the microarray chips was performed at the Microarray Department (MAD) of the University of Amsterdam (Amsterdam, The Netherlands) using the standard Agilent protocol.
P-GSE42847-5
labeling
Amino Allyl modified aRNA was synthesized in one amplification round from 1 ug of total RNA using the Amino Allyl MessageAmp II aRNA Amplification Kit (Ambion). Subsequently, 6 ug of Amino Allyl modified aRNA was used for coupling of monoreactive Cy3 or Cy5 dye (GE Healthcare) and column purified.
P-GSE42847-4
nucleic acid library construction protocol
Embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at -80C. Embryos were homogenized in 1 ml of TRIZOL Reagent (Invitrogen) and subsequently total RNA was extracted according to the manufacturer’s instructions. The RNA samples were incubated for 20 min at 37C with 10 units of DNaseI (Roche Applied Science) to remove residual genomic DNA prior to purification using the RNeasy MinElute Cleanup kit (Qiagen) according to the RNA clean up protocol. The integrity of the RNA was confirmed by Lab-on-chip analysis using the 2100 Bioanalyzer (Agilent Technologies).
P-GSE42847-2
specified_biomaterial_action
Zebrafish embryos were micro-injected into the yolk (2hpf) with (1) 20 CFU of S. epidermdis O-47 mCherry bacteria suspended in PVP (Polyvinylpyrrolidone), (2) mock-injected with PVP as a control, (3) Needle insertion as control, (4) Non-injected as a control. At 8 hpf (6 h post infection), 32 hpf (30 h post infection), 56 hpf (54 h post infection), 80 hpf (78 h post infection), 104 hpf (102 h post infection) or 128 hpf (126 h post infection) twenty embryos per treatment group were snap-frozen in liquid nitrogen, and total RNA was isolated using TRIZOL reagent.
P-GSE42847-3
grow
Zebrafish were handled in compliance with the local animal welfare regulations and maintained according to standard protocols (http://ZFIN.org). Embryos were grown at 28.5C in egg water (60µg/ml Instant Ocean sea salts).
P-GSE42847-12
feature_extraction
Base calling was done by the Illumina HCS version 1.15.1 Sequence reads were quality trimmed using the quality_trim module in the CLCbio Assembly Cell v4.0.6. Filtered reads were mapped to Ensembl transcripts (Zv9_63) using the ref_assemle_short module in the CLCbio Assembly Cell v4.0.6. Accumulation of transcripts to Ensembl genes was done by first converting the mapping files to a table with the assembly_table module in the CLCbio Assembly Cell v4.0.6. Secondly, a custom script was used that sums all reads belonging to the same gene. Non-uniquely mapped reads were divided between genes according to their ratio of uniquely mapped reads. Finally, read counts of transcripts belonging to the same gene were summed to obtain count data at Ensembl gene level. Fold-change and differential expression significance values were calculated from gene level read counts using the DESeq package (version 1.8.3) available in Bioconductor (version 2.10). Genome_build: Zv9_toplevel Supplementary_files_format_and_content: tab-delimited text files include RPKM values for each Sample
P-GSE42847-11
nucleic acid library construction protocol
Embryos for RNA isolation were snap frozen in liquid nitrogen and subsequently stored at -80°C. Embryos were homogenized in 1 ml of TRIZOL® Reagent (Invitrogen) and subsequently total RNA was extracted according to the manufacturer’s instructions. The RNA samples were incubated for 20 min at 37° with 10 units of DNaseI (Roche Applied Science) to remove residual genomic DNA prior to purification using the RNeasy MinElute Cleanup kit (Qiagen) according to the RNA clean up protocol. The integrity of the RNA was confirmed by Lab-on-chip analysis using the 2100 Bioanalyzer (Agilent Technologies). RNA libraries were prepared for sequencing using standard Illumina protocols
P-GSE42847-10
grow
Zebrafish were handled in compliance with the local animal welfare regulations and maintained according to standard protocols (http://ZFIN.org). Embryos were grown at 28,5°C in egg water (60µg/ml Instant Ocean sea salts).
P-GSE42847-9
specified_biomaterial_action
Zebrafish embryos were micro-injected into the yolk (2hpf) with 20 CFU of S. epidermdis O-47 mCherry bacteria suspended in PVP (Polyvinylpyrrolidone), or Non-injected as a control. At 5 days post injection 100-200 embryos per treatment group were snap-frozen in liquid nitrogen, and total RNA was isolated using TRIZOL reagent.