RNA-seq from ENCODE/LICR

Using RNA-Seq (Mortazavi et al., 2008), high-resolution genome-wide maps of the mouse transcriptome across multiple mouse (C57Bl/6) tissues and primary cells were generated. Cells were grown according to the approved ENCODE cell culture protocols (http://genome.ucsc.edu/ENCODE/protocols/cell/mouse). RNA-Seq RNA samples from tissues and primary cells were extracted from Trizol according to protocol (Invitrogen). PolyA+ RNA was purified with the Dynabeads mRNA purification kit (Invitrogen). The mRNA libraries were prepared for strand-specific sequencing as described previously (Parkhomchuk et al., 2009). Sequencing and Analysis Samples were sequenced on Illumina Genome Analyzer II, Genome Analyzer IIx and HiSeq 2000 platforms for 36 cycles. Image analysis, base calling and alignment to the mouse genome version mm9 were performed using Illumina's RTA. Alignment to the mouse genome was performed using TopHat (Trapnell et al., 2009). Wig files were generated by TopHat and expression levels were calculated with Cufflinks (Trapnell et al., 2010).本研究基于RNA测序技术（RNA-Seq，Mortazavi等，2008），构建了覆盖多种C57Bl/6品系小鼠组织与原代细胞的高分辨率全基因组转录组图谱。细胞培养严格遵循ENCODE官方批准的细胞培养规程（详见http://genome.ucsc.edu/ENCODE/protocols/cell/mouse）。组织与原代细胞的RNA-Seq测序样本总RNA均按照Invitrogen公司提供的Trizol试剂操作流程提取；随后采用Invitrogen公司的Dynabeads mRNA纯化试剂盒富集得到聚腺苷酸RNA（PolyA+ RNA）。按照此前报道的方法（Parkhomchuk等，2009）构建链特异性测序所需的mRNA文库。测序与数据分析阶段：样本分别在Illumina Genome Analyzer II、Genome Analyzer IIx及HiSeq 2000测序平台上进行36个循环的测序。图像分析、碱基识别以及比对至小鼠基因组版本mm9的步骤均通过Illumina的RTA软件完成；小鼠基因组比对分析则借助TopHat工具完成（Trapnell等，2009）。通过TopHat生成WIG格式文件（Wig files），并利用Cufflinks计算基因表达水平（Trapnell等，2010）。