Computational protocol: DNA methylation alters transcriptional rates of differentially expressed genes and contributes to pathophysiology in mice fed a high fat diet

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Protocol publication

[…] RNA concentration and integrity were determined by Bioanalyzer using the RNA Nano Kit (Agilent Technologies, Santa Clara, CA). RNA-seq libraries were prepared using the TruSeq RNA Sample Prep Kit (Illumina, San Diego, CA) per the manufacturer's protocol. Briefly, mRNA was purified from total RNA using oligo dT magnetic beads, fragmented, and then reverse transcribed via random hexamers and SuperScript II Reverse Transcriptase (Invitrogen, Carlsbad, CA). Second strand cDNA was synthesized using a proprietary master mix. The double stranded cDNA was then blunt ended, the 3′ ends were adenylated, and indexed Illumina adaptors were ligated. DNA fragments were enriched by PCR using the following conditions: initial denaturation of 98 °C for 30 s, 15 cycles of 98 °C for 10 s, 60 °C for 30 s, and 72 °C for 30 s, followed by a final extension of 72 °C for 5 min. RNA-seq libraries were visualized by Bioanalyzer (Agilent) and quantified by qPCR. Libraries were bar-coded and pooled by quantity and sequenced via 50 bp single reads on a HiSeq2000 (Illumina). Reads were trimmed to include only bases 9–57 for quality purposes. Alignment was done to mm9 using Tophat with the Bowtie 1 option , . The number of reads intersecting with each gene was calculated, one was added, and counts were log2 transformed. DESeq was used for differential expression testing . [...] Liver tissue was homogenized via Tissue Lyser II (Qiagen) and DNA was extracted via DNeasy Blood and Tissue Kit (Qiagen) per manufacturer's instructions. DNA libraries were prepared as per the SureSelectXT Methyl-Seq Target Enrichment System Protocol (Agilent) with a 9 cycle PCR post bisulfite conversion and a 6 cycle indexing PCR. SureSelect Mouse All Exon V1 52 Mbp was used for capture. Libraries were sequenced on a Hiseq2000 (Illumina) using 100 bp paired end reads. Quality and adaptor trimming were done using Trim Galore and alignment was performed using Bismark. Duplicates were removed. MethylSig was used for differential methylation analysis , . The GEO accession number for this study is GSE85425. […]

Pipeline specifications

Software tools Trim Galore!, Bismark, methylSig
Application BS-seq analysis
Organisms Mus musculus
Diseases Fatty Liver, Metabolic Diseases