RNA-seq data of ox-LDL treated rabbit VSMCs

RNA
extraction and preparation for RNA-Sequencing
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.
Quality
assessment, genome mapping, and transcriptome assembly
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
(Oryctolagus cuniculus) 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.
LncRNA
identification
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).
Expression
analysis

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 P
value <0.05 was defined as significant difference between control group and
ox-LDL treated group.