RNA seq of cardiomyocytes (CMCs) and vascular smooth muscle cells (SMCs) of CMS/SMC-specific TEAD1-KO and control mice

SM22a-Cre+/TEAD1F/W male mice were bred with TEAD1F/F/mTmG+/+ female mice to generate cardiomyocyte (CMC) / vascular smooth muscle cell (VSMC)-specific TEAD1 deletion with GFP tag. GFP positive heterozygous control and TEAD1 KO cardiac and DA tissues were dissected under a fluorescent stereoscope. Subsequently the GFP positive CMCs and VSMCs were dissociated by enzymatic digestion and collected by fluorescence activated cell sorting (FACS). Total RNA was then extracted from the purified GFP positive cells using RNeasy Micro Kit (Qiagen) and subjected to whole transcriptome RNA-seq analysis. Differential gene expression analysis was performed at the Genome Technology Access Center at Washington University. Following amplification of the input RNA by Sigma SeqPlex RNA amplification kit, library was prepared and single-end sequencing in 50-bp length was performed on a HiSeq 3000 system (Illumina).

将SM22a-Cre+/TEAD1F/W雄性小鼠与TEAD1F/F/mTmG+/+雌性小鼠交配，以构建携带绿色荧光蛋白（green fluorescent protein, GFP）标签的心肌细胞（cardiomyocyte, CMC）与血管平滑肌细胞（vascular smooth muscle cell, VSMC）特异性TEAD1敲除模型。在体式荧光显微镜下解剖获取GFP阳性的杂合对照小鼠及TEAD1敲除小鼠的心脏与动脉导管（ductus arteriosus, DA）组织。随后通过酶解法解离GFP阳性的心肌细胞与血管平滑肌细胞，并通过荧光激活细胞分选（fluorescence activated cell sorting, FACS）收集目标细胞。使用RNeasy Micro试剂盒（Qiagen）从纯化后的GFP阳性细胞中提取总RNA，开展全转录组RNA测序（RNA-seq）分析。差异基因表达分析工作由华盛顿大学基因组技术访问中心（Genome Technology Access Center）完成。使用Sigma SeqPlex RNA扩增试剂盒对起始RNA进行扩增后，构建测序文库，并在HiSeq 3000测序系统（Illumina）上完成50bp单端测序。