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

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.

研究目的：本研究旨在鉴定经地塞米松处理的SETDB2缺陷型小鼠原代肝细胞（MPH）与对照小鼠原代肝细胞之间的差异表达基因。实验方法：分别采用shControl或shSETDB2腺病毒处理小鼠原代肝细胞（每组3只小鼠的样本混合），依照制造商说明书，使用TRIzol法（Life Technologies公司）提取总RNA。总RNA经DNase处理后，采用生物分析仪（Biolanalyzer，安捷伦公司）评估RNA完整性。RNA测序由位于美国佛罗里达州奥兰多市莱克诺纳的SBP基因组学核心实验室（SBP Genomics Core Facility）依托Illumina HiSeq平台完成，测序参数如下：双端读长100 BP；产出读段（reads）数为3亿条。RNA测序分析采用此前报道的Tophat+Cufflinks流程（Haas et al., 2013; Trapnell et al., 2012）。以2倍表达差异阈值、p值小于0.05以及FPKM（每百万比对读段对应每千碱基转录本的片段数，fragments per kilobase of transcript per million mapped reads）阈值为1，鉴定shSETDB2与shControl处理的小鼠原代肝细胞之间的差异表达基因。使用Tophat（Trapnell et al., 2009）将shSETDB2与shControl组的RNA-seq读段比对至mm10参考基因组，随后通过Cufflinks对比对结果进行进一步分析，最终输出差异基因列表。整体实验设计：采用Illumina HiSeq平台进行深度测序，获取shControl与shSETDB2腺病毒感染的小鼠原代肝细胞的mRNA表达谱（每组n=3只小鼠）。