Sample 15_p
C57BL/6J mice (6 weeks old, n=30) were euthanized, and the heart was perfused through the apex with sterile HBSS. Aortas (a pool of 15 per experiment) were dissected and the perivascular tissue was removed, followed by two washes in ice-cold HBSS.  To facilitate removal of the adventitial layer, aortas were placed in 2 mg/ml collagenase II enzyme solution for 10 min at 37°C followed by washing in DMEM/F12 media. The adventitia was stripped off, and the aortas were opened longitudinally and washed in DMEM/F12 to remove blood clots. The endothelial layer was then removed by gently scraping with fine forceps followed by washing in DMEM/F12 and mincing of the remaining medial layer into small pieces with scissors, which were incubated for second digestion with an enzymatic solution (2 mg/ml collagenase II, 1 mg/ml soybean trypsin inhibitor, 0.75 U/ml elastase, 1% penicillin/streptomycin in HBSS) for 1 h at 37°C with shaking. 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 ~80% confluency (P4) were serum starved for 24 h, before the assays. We performed two different experiments. In experiment A, control (transfected with 10 μM control siRNA) and Ikbkb-deficient cells (transfected with 10 μM Ikbkb siRNA) were left untreated or exposed to water-soluble cholesterol (Sigma-Aldrich, C4951, final concentration 10 μg/mL) for 72 hours. In experiment B, control and Ikbkb-deficient cells were left untreated or exposed to human TNF (ab259410, final concentration 10 ng/ml in PBS containing 0.1% BSA) for 24 hours. For each condition, we obtained RNA from a pool of 3 wells and we used 4 replicates). Transfection of siRNAs in mouse aortic SMCs was carried out using Opti-MEM (Thermo Fisher) and Lipofectamine RNAiMAX (Invitrogen) for 18 h on two consecutive days. To knockdown Ikbkb, we used a pool of two independent mouse siRNAs (Qiagen FlexiTube siRNA) with the following target sequences: CAGGTTATCATAGGCAGGTAT and CTGCTGTTTCTGGTCCTTTAA. As control siRNA we used the AllStars negative control siRNA (Qiagen, cat. no. 1027280). All siRNA were from Qiagen.   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.