HUVECs treated with PBS as control (Rep1)
Total RNA was isolated using the Trizol Reagent (Invitrogen Life Technologies), and the concentration, quality, and integrity were determined using a NanoDrop spectrophotometer (Thermo Scientific). Ribosomal RNA (rRNA) was removed using the Epicentre Ribo-Zero rRNA Removal Kit (Human/Mouse/Rat). Linear RNA was removed using RNase R (Epicentre, Inc.), and residual poly-A RNA was depleted using poly-T oligo-attached magnetic beads. RNA fragmentation was performed using divalent cations under elevated temperature in an Illumina proprietary fragmentation buffer. First-strand cDNA synthesis was carried out using random oligonucleotides as primers, followed by RNA strand degradation with RNase H and second-strand cDNA synthesis using DNA polymerase I with dNTPs where dUTP was substituted for dTTP. Double-stranded cDNA was purified, end-repaired, and an "A" base was added to the 3' ends before ligation of Illumina sequencing adapters. USER enzyme (NEB, USA) was used to degrade the second strand of cDNA containing U. Library fragments of 400-500 bp were purified using the AMPure XP system (Beckman Coulter, Beverly, CA, USA). DNA fragments with ligated adapter molecules were enriched using Illumina PCR Primer Cocktail in a 15-cycle PCR reaction. The final libraries were quantified using the Agilent high sensitivity DNA assay on a Bioanalyzer 2100 system (Agilent) and sequenced on the Illumina NovaSeq 6000 platform by Shanghai Personal Biotechnology Co., Ltd.