RNA-seq of dexamethasone-treated mouse primary hepatocytes infected with shControl or shSETDB2 adenovirus. Mus musculus

Purpose: The goal of this study is to identify differentially expressed genes in SETDB2 deficient mouse primary hepatocytes (MPH) treated with dexamethasone when compared to control MPH Methods: Total RNA from MPH treated with either shControl or shSETDB2 adenovirus (pool of 3 mice per group) was isolated by the TRIzol method (Life Technologies) according to manufacturer’s instructions. Total RNA was DNase-treated and RNA integrity was evaluated by Biolanalyzer (Agilent). RNA sequencing was performed by the SBP Genomics Core Facility in Lake Nona (Orlando, FL) using the Illumina HiSeq platform and the following parameters: paired-end read length: 100 BP; number of output reads: 300m reads. The Tophat+Cufflinks pipeline was used for the RNA-seq analysis as described previously (Haas et al., 2013; Trapnell et al., 2012). Differentially expressed genes in shSETDB2 vs. shControl MPH were determined using a 2-fold cutoff, p-value of less than 0.05, and FPKM (fragments per kilobase of transcript per million mapped reads) cutoff of 1. The shSETDB2 and shControl RNA-seq reads were mapped to the mm10 genome using Tophat (Trapnell et al., 2009). The mapping results were further analyzed by Cufflinks, which outputs the final differential gene list. Overall design: mRNA profiles of shControl and shSETDB2 adenoviral infected mouse primary hepatocyte (n=3 mice) were generated by deep sequencing using Illumina HiSeq platform.