RNA-seq Analysis of SREBP2 Overexpression

Total RNA was isolated from HUVECs infected with adenovirus SREBP2 (Ad-SREBP2) or Ad-null (empty vector). mRNA was isolated using mirVana mRNA isolation kit (Thermo Fisher Scientific). Standard Illumina protocols were used to construct the RNA libraries and sequencing. Analysis was performed using base calling and quality scoring by using Real-Time Analysis version 2 (RTA v2) on the NextSeq 500 system. Data were demultiplexed and converted to FASTQ files using Bcl2fastq conversion software v1.8.4. Sequence reads were trimmed of their adaptor sequences and masked for low complexity or low-quality sequence. Reads were mapped to the h19 genome using tophat v2.0.14 (55 (link)). Data were normalized to reads per kilobase of transcript, per million mapped reads (RPKM) using cufflinks v2.2.1 to assemble transcripts and estimate mRNA abundance (Supplemental Figure 4) (56 (link), 57 (link)). Data are available at NCBI’s Gene Expression Omnibus database (GSE121782).