DOI: 10.6084/m9.figshare.5446705.v1
title: RNA-seq data of ox-LDL treated rabbit VSMCs

description: <b>RNA extraction and preparation for RNA-Sequencing</b>Total RNA of each sample was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA). The NanoPhotometer spectrophotometer (IMPLEN, Westlake Village, CA), the Qubit RNA Assay Kit in Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA), and the RNA Nano 6000 Assay Kit of Bioanalyzer 2100 System (Agilent Technologies, Santa Clara, CA) ware utilized to check the purity, concentration, and integrity of RNA respectively.In present study, eight complementary DNA (cDNA) libraries were constructed in total, i.e., four for rabbit VSMCs as control group and another four for ox-LDL-treated VSMCs. 3 µg RNA per sample was used as input material. Ribosomal RNA was removed by Epicentre Ribo-Zero rRNA Removal Kit (Epicentre, Madison, WI) firstly. Residue of rRNA-free was cleaned through ethanol precipitation. Second, sequencing libraries were generated using NEB-Next Ultra Directional RNA Library Prep Kit for Illumina (NEB, Ipswich, MA).First-strand cDNA were synthesized through M-MuLV Reverse Transcriptase (RNaseH-) plus random hexamer primer. Then second-strand cDNA was synthesized subsequently using RNase H and DNA Polymerase I were utilized for synthesis of second-strand cDNA. The 3′ends of DNA fragments were adenylated, then NEBNext Adaptor with hair-pin loop structure were prepared for hybridization. AMPure XP System (Beckman Coulter, Beverly, MA) was used to purify the library fragments with preference of 150-200 bp in length. Finally, the library quality was assessed by Agilent Bioanalyzer 2100 System. Clustering of samples was performed on cBot Cluster Generation System though TruSeq PE Cluster Kit v3-cBot-HS (Illumina). Then libraries were sequenced at the Novogene Bioinformatics Institute (Beijing, China) on Illumina HiSeq 4000 Platform, and paired-end reads with 150 bp were generated by Illumina HiSeq 2500 Platform.<b>Quality assessment, genome mapping, and transcriptome assembly </b>Clean reads were screen out from raw data by removing reads with adapter, poly-N or low quality. Then, calculation were performed to assess Q20, Q30, and GC contents of the clean reads. Clean reads of high-quality were used for subsequent analyses. The rabbit (<i>Oryctolagus cuniculus</i>) genome (ftp://ftp.ensembl.org/pub/release-88/embl/oryctolagus_cuniculus/) was used as reference genome for reads mapping through TopHat v2.0.9. Cufflinks v2.1.1 was used for mapped reads assembly of each sample. <b>LncRNA identification </b>All successfully assembled reads were combined by Cuffcompare software. Then transcripts shorter than 200 bp in length or less than two exons was removed. Reads coverage was calculated by Cufflinks v2.1.1 and transcripts less than three reads coverage were abandoned. Comparison between transcripts and reference rabbit lncRNAs was performed by Cuffcompare software. RNA transcripts like tRNA, rRNA, snoRNA, snRNA, pre-miRNA, and pseudogenes were also detected and discarded. Then, transcripts of mRNA were gained from blasting with annotated rabbit mRNA. Software CNCI, CPC, PFAM, and phyloCSF were utilized to screen out lncRNA by assessing the coding potential of transcripts. The final results contained lncRNAs which classified into three subtypes as lincRNA, intronic lncRNA, and antisense lncRNA through class_code (http://coLetrapnelllabio/cufflinks/cuffcompare/index.Html#transfrag-classcodes).<b>Expression analysis</b> The expression levels of lncRNAs and mRNAs in each sample were evaluated using Cuffdiff (v2.1.1). Fragments per kilo-base of exon per million fragments (FPKM) mapped of the transcripts was calculated using Cuffdiff (v2.1.1). Then results were used for evaluating the expression levels of lncRNAs and mRNAs in each sample. Expression of transcripts with <i>P</i> value &lt;0.05 was defined as significant difference between control group and ox-LDL treated group.