GSM9405367: SMC, Foam Cell Media; Homo sapiens; RNA-Seq
Human primary coronary artery smooth muscle cells (SMC) (Lot# 20TL266549, Cat# CC-2583, Lonza) were cultured in completed SmGM-2 growth medium (#CC-3182, Lonza). Foam cell conditioned medium was collected from cultures of THP-1-derived foam cells as previously described, with supernatant retrieval following centrifugation at 150 g for 5 minutes and filtration through a 0.22 μm membrane filter (#GSWP02500, Millipore). On day 0, the medium swap group was plated in 6-well plates (#140675, Thermo Fisher Scientific) at a density of 1 x 105 cells per well in SmGM basal medium containing 5% FBS (BM-FBS medium). On day 4 and day 8, the medium was replaced with fresh BM-FBS medium containing 50% foam cell conditioned medium. The control group was plated at the same density in completed SmGM-2 growth medium three days before harvest. On day 12, SMCs were detached by adding 1 mL of 0.05% trypsin-EDTA (#25300-054, Gibco) per well and incubating at 37°C for 3 minutes, then neutralized with 10 mL of BM-FBS medium. Viable cells were counted using trypan blue solution (#15250061, Gibco). The cell suspension was subsequently filtered through a 100 µm nylon cell strainer (#352360, Falcon) to remove clumps, resulting in a uniform single-cell suspension for further analysis. Library preparation was conducted with Chromium GEM-X Single Cell 3' Reagent Kits v4 (10x Genomics). Libraries were sequenced on NovaSeq X Plus. The initial 10x scRNA-seq dataset was processed using the Cell Ranger pipeline (v.8.0.0). The workflow included demultiplexing the bcl files with cellranger mkfastq, followed by running cellranger count using the pre-built Cell Ranger reference GRCh38-2020-A from 10x Genomics.