DNBSEQ-G400 paired end sequencing; Cholesterol and TNF-induced transcriptomic changes in Mus musculus aortic smooth muscle cells
Cholesterol and TNF-induced transcriptomic changes in Mus musculus aortic smooth muscle cells
C57BL/6 mice (8-10 weeks old) were euthanized and the heart was perfused through the apex with sterile HBSS. Aortas (pool of 5) were dissected out and the perivascular tissue removed followed by washing twice in ice cold HBSS. To facilitate the removal of the adventitial layer, aortas were placed in an enzyme solution for 10 min at 37°C (2 mg/ml collagenase II, 1 mg/ml soybean trypsin inhibitor, 0.75 U/ml elastase, 1% penicillin/streptomycin in HBSS) followed by washing in DMEM/F12 medium. Adventitia was stripped off and the aortas were opened longitudinally and washed in DMEM/F12 medium to removed blood clots. The endothelial cell layer was then removed by gently scraping with fine forceps followed by washing in DMEM/F12 medium and mincing of the remaining medial layer into small pieces with scissors, which were incubated for a second digestion with the enzyme solution for 1 h at 37°C. Digested aortas were rigorously pipetted up and down several times and the cell suspension was filtered through a 70-μm cell strainer before undergoing 3 cycles of centrifugation (1000 rpm, 5 min, room temperature) and washing in DMEM/F12 medium to remove the enzyme solution. The cell pellet was finally resuspended in DMEM/F12 medium containing 20% fetal bovine serum (FBS) and 1% penicillin/streptomycin, and SMCs were seeded into collagen type-I-coated 24-well culture plates. Two days later, cell medium was changed to DMEM/F12 medium containing 10% FBS and 1% penicillin/streptomycin. Aortic SMCs at a confluency of ~80% (P4) were serum starved for 24 h and culture wells were allocated into 4 groups (4 wells per group). Group 1 was left untreated for 72 hours (control). Group 2 was treated with human TNF protein (final concentration 10 ng/µL in PBS containing 0.1% BSA, R&D systems, 210-TA-020) for the final 24 hours. Group 3 was treated with water-soluble cholesterol (final concentration 10 μg/mL, Sigma-Aldrich, C4951) for 72 hours. In Group 4, cholesterol and TNF treatment as described above was combined. RNA was isolated from wells using the NucleoSpin RNA Plus Kit (Macherey-Nagel, 790984) RNA sequencing was performed by the BGI sequencing service. All RNA samples passed quality control (Agilent 4200 electrophoresis system). For the library preparation non-stranded and polyA-selected mRNA were used. Non-stranded DNA libraries were produced.