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. 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.