RNA-Seq of Homo sapiens: adult aortic smooth muscle cell
RNA integrity was assessed using the RNA Nano 6000 Assay Kit with the Bioanalyzer 2100 system (Agilent Technologies). For library preparation, 1 g of RNA per sample was used. mRNA was purified with poly-T oligo-attached magnetic beads, fragmented with divalent cations at elevated temperature, and synthesized into first strand cDNA using random hexamer primer and M-MuLV Reverse Transcriptase. Second strand cDNA was synthesized using DNA Polymerase I and RNase H, and overhangs were converted into blunt ends. After adenylation of 3' ends, adaptors with hairpin loops were ligated. cDNA fragments of 370-420 bp were selected using the AMPure XP system (Beckman Coulter). PCR amplification was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers, and Index Primer, followed by purification and quality assessment with the AMPure XP system and Agilent Bioanalyzer 2100 system. Clustering was performed on a cBot Cluster Generation System with TruSeq PE Cluster Kit v3-cBot-HS (Illumina), and sequencing on an Illumina Novaseq platform generated 150 bp paired-end reads.