RNA-Seq of Homo sapiens: male aorta
RNA from total samples was isolated and purified using TRIzol (thermofisher, 15596018) according to the protocol provided by the manufacturer. The amount and purity of total RNA was then quality controlled with a NanoDrop ND-1000 (NanoDrop, Wilmington, DE, USA) and the integrity of the RNA was examined by a Bioanalyzer 2100 (Agilent, CA, USA); a concentration of >50 ng/L, a RIN value of >7.0, and a total RNA >1 g were sufficient for downstream experiments. total RNA >1 g were sufficient for downstream experiments. The mRNA with PolyA (polyadenylate) was specifically captured by two rounds of purification using oligo(dT) magnetic beads (Dynabeads Oligo (dT), cat.25-61005, Thermo Fisher, USA). The captured mRNAs were fragmented using the Magnesium Ion Interrupter Kit (NEBNextR Magnesium RNA Fragmentation Module, cat. E6150S, USA) under high temperature conditions, 94C, 5-7 min. The fragmented RNA was passed through reverse transcriptase (Invitrogen SuperScriptTM II Reverse Transcriptase, cat.1896649, CA, USA) to synthesise cDNA. cDNA was then synthesised using E. coli DNA polymerase I (NEB, cat.m0209, USA) with RNase H (cat.m0209, USA). (NEB, cat.m0209, USA) and RNase H (NEB, cat.m0297, USA) for two-stranded synthesis to convert these composite double strands of DNA and RNA into DNA duplexes, while doping the duplexes with dUTP Solution (Thermo Fisher, cat.R0133, CA, USA) to complement the ends of double-stranded DNA to flat ends. An A base was then added to each of its ends to enable it to be ligated to a junction with a T base at the end, and its fragment size was screened and purified using magnetic beads. The second strand was digested with UDG enzyme (NEB, cat.m0280, MA, US), and the fragment size of 300 bp  50 bp was formed by PCR-pre-denaturation with a 95C hold for 3 min, denaturation at 98C for a total of 8 cycles of 15 s each, annealing to 60C with a hold of 15 s, extension at 72C for 30 s, and a final extension with a hold at 72C for 5 min. 50bp (strand-specific library). Finally, we used illumina NovaseqTM 6000 to perform bipartite sequencing according to the standard operation, and the sequencing mode was PE150