Computational protocol: Defining baseline epigenetic landscapes in the rat liver

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Protocol publication

[…] We utilized a number of published microarray gene expression datasets for adult male and female Wistar rats (each n = 4) []. RNA was extracted from snap-frozen livers by grinding in RTL lysis buffer (Qiagen, MD, USA) prior to isolation using RNAeasy spin columns (Qiagen). Starting from 300 ng total RNA, biotin-labeled cRNA samples for hybridization on Affymetrix RaGene_2.0_ST arrays were prepared according to the protocol supplied with the GeneChip® WT PLUS Reagent Kit for Affymetrix GeneChip Whole Transcript (WT) Expression Arrays (P/N 703174 Rev. 2, Affymetrix Inc, CA, USA). 3 μg fragmented ssDNA was hybridized for 16 h at 45°C. The microarrays were washed and stained with streptavidin-phycoerythrin (Molecular Probes). The fluorescent images of the GeneChips were captured with the Affymetrix GeneChip Scanner 3000. Microarray data were processed with the R/Bioconductor package ‘affy’ version 1.44.1 modified by Brainarray lab for Gene ST support. Brainarray Custom CDF ragene20st version 19.0.0 was used for summarization. Raw data were normalized using RMA as implemented in the ‘affy’ package. To determine tissue-specific and housekeeping rat gene sets (), we used a number of datasets from a large-scale rat RNAseq project [] (male liver: GSM1328657, female liver: GSM1328641, male lung: GSM1328613, female lung: GSM1328598, male kidney: GSM1328581, female kidney: GSM1328565, male heart: GSM1328549 and female heart: GSM1328533) and selected the 250 top-induced genes unique to each tissue. The top 250 housekeeping genes were selected as exhibiting similarly high-expression levels in all datasets. [...] Functional analysis of gene sets associated with differentially modified promoter or gene bodies was carried out using the GO TERM BP FAT feature on the DAVID functional annotation tool database [].Accession codesWe are currently in the process of uploading all raw and processed sequencing datasets at the Gene accession omnibus (GEO). Accession pending. Wistar male and female gene expression datasets can be found at GEO under the accession number GSE68128 and include datasets GSM1664281, GSM1664282, GSM1664283, GSM1664284, GSM1664289, GSM1664290, GSM1664291 and GSM1664292. Fischer 344 Rat RNAseq datasets are found at GEO under accession numbers GSM1328657, GSM1328641, GSM1328613, GSM1328598, GSM1328581, GSM1328565, GSM1328549 and GSM1328533. […]

Pipeline specifications

Software tools affy, DAVID
Application RNA-seq analysis
Organisms Rattus norvegicus, Homo sapiens
Chemicals 5-Methylcytosine