RNA-seq Analysis of Ox-LDL-treated Rabbit VSMCs

Total RNA of each sample was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The NanoPhotometer spectrophotometer (IMPLEN, Westlake Village, CA, USA), the Qubit RNA Assay Kit in Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA), and the RNA Nano 6000 Assay Kit of Bioanalyzer 2100 System (Agilent Technologies, Santa Clara, CA, USA) ware utilized to check the purity, concentration, and integrity of RNA respectively. Eight cDNA libraries in total were constructed from ox-LDL-treated rabbit VSMCs (n = 4) and control group (n = 4). Sequencing libraries were generated using NEB-Next Ultra Directional RNA Library Prep Kit for Illumina (NEB, Ipswich, MA, USA) followed by library fragments purification and quality assessment. Finally libraries were sequenced on Illumina HiSeq 4,000 Platform, and paired-end reads with 150 bp were generated by Illumina HiSeq 2,500 Platform. We screened out clean reads of high-quality from raw data (66.1 GB, SRA accession ID: SRP124805) for subsequent analyses. The rabbit (Oryctolagus cuniculus) genome (OryCun2.0 in the NCBI Assembly database) was used as reference genome for reads mapping through TopHat v2.0.9 (Kim et al., 2013 (link); Trapnell, Pachter & Salzberg, 2009 (link)). Cufflinks v2.1.1 was used for mapped reads assembly of each sample (Trapnell et al., 2010 (link)).