RNA-Seq analysis, transcriptome assembly and gene expression profile analysis for vascular smooth muscle cells with up- or downregulation of CKLF1

To understand the role of Cklf1 in the proliferation of vascular smooth muscle cells (VSMC), we quantified the whole genome transcriptomes of VSMC that were treated by Cklf1 adenovirus (n=3) or control (n=3) for 24 h using RNA-seq. In total, we obtained over 82 million clean reads from each library after trimming adaptor sequences, low quality reads and multiple mapped reads. The clean reads were mapped to 29330 genes annotated in the rat reference genome (Rattus_norvegicus 6.0). We then identified differentially expressed genes (DEGs) between Cklf1-associated adenovirus and controls by comparing RNA-Seq data between the two groups. A total of 238 DEGs for Ad-GFP vs. Ad-Cklf1 and 468 DEGs for shRNA Scramble vs. shRNA Cklf1 were defined using the thresholds of FDR = 0.05, difference ratio of FPKM (fragments per kilobase of exon model per million mapped fragments) = 2 and diverge probability = 0.8. The increased CKLF1 resulted in 34 up-regulated and 204 down-regulated genes while knockdown of Cklf1 led to 358 up-regulated and 110 down-regulated genes. Overall design: Vascular smooth muscle cells were obtained from the media of normal thoracic aorta of male Sprague-Dailey rats (100 ± 10 g) using primary explant technique. Cells were incubated with Cklf1 adenovirus (shRNA CKLF1 or CKLF1) and control (shRNA scramble or none) overnight and cultured as described in growth protocol. Total RNA were extracted and generated by deep sequencing, in triplicate, using Illumina X Ten sequencer.

为阐明Cklf1在血管平滑肌细胞（vascular smooth muscle cells, VSMC）增殖中的作用，本研究采用RNA测序技术，对经Cklf1腺病毒（adenovirus）处理（3个生物学重复）或对照处理（3个生物学重复）24小时的VSMC进行全基因组转录组定量分析。经修剪接头序列、低质量读数及多映射读数后，每个文库均获得超过8200万条清洁读数（clean reads）。清洁读数被比对至大鼠参考基因组（Rattus_norvegicus 6.0）注释的29330个基因。随后通过比较两组的RNA测序数据，鉴定Cklf1相关腺病毒与对照组之间的差异表达基因（differentially expressed genes, DEGs）。基于错误发现率（false discovery rate, FDR）=0.05、FPKM（每百万映射片段中外显子模型每千碱基片段数，fragments per kilobase of exon model per million mapped fragments, FPKM）差异倍数=2及发散概率=0.8的阈值，共鉴定得到Ad-GFP与Ad-Cklf1组的238个DEGs，以及shRNA Scramble与shRNA Cklf1组的468个DEGs。过表达CKLF1可导致34个基因上调、204个基因下调；而敲低Cklf1则可引起358个基因上调、110个基因下调。本研究整体实验设计如下：采用原代组织块贴壁法，从体重100±10 g的雄性Sprague-Dailey大鼠正常胸主动脉中膜分离得到血管平滑肌细胞。将细胞分别与携带shRNA CKLF1或CKLF1过表达序列的Cklf1腺病毒及对照（携带shRNA scramble或无处理）孵育过夜，随后按照标准生长培养方案进行培养。提取总RNA并进行深度测序，每组设置3次生物学重复，采用Illumina X Ten测序仪完成测序。