subexperiment: Growth arrest: MMC coriell_id: AG11546 cell_line: Adult_vascular_smooth_muscle_cell_1 donor_age: 19 years donor_sex: male culture_oxygen: Ambient culture_agent: MMC culture_pctfbs: Full expression_vector: NA subculture: 3 population_doublings: 30.47800398 days_in_culture: 18 batch_date: 02.20.20 growth protocol: Vascular smooth muscle cells were maintained in Medium 199 with 1X GlutaMAX, 0.02mg/ml endothelial cell growth supplement, 0.05 mg/ml sodium heparin, and 10% v/v fetal bovine serum on plates pre-coated with gelatin. Triplicate cultures derived from the same parent plate or vial obtained from Coriell were maintained in parallel through replicative senescence, which was defined in this study as drastically slowed growth (inability to reach near-confluence at 14 days after previous passage) or viable fraction of cells falling below 60%. Passaging occurred as cells became approximately 90% confluent. treatment protocol: Primary cells were reintroduced into culture from cryopreserved early-passage cells. Duplicate growth-arrest subcultures were derived from the initial recovered plate. Cells were treated with D intercalating agent mitomycin C reconstituted in DMSO at a fil concentration of 10ug/ml. Cells were incubated for 3 hours at 37°C before media containing MMC was removed, cells rinsed with PBS, and basal media replaced. Cells were collected on days 18 and 25.
Extracted molecule
genomic DNA
Extraction protocol
Frozen cell pellets were thawed and lysed using QIAshredder spin columns (Qiagen). Genomic DNA was extracted from each sample using the AllPrep DNA/RNA Mini Kit (Qiagen), then stored at -80°C before analysis.
Label
Cy3 and Cy5
Label protocol
Cy3 and Cy5
Hybridization protocol
Bisulfite-converted DNA was amplified, fragmented, and hybridized to Illumina Infinium Human Methylation850k Beadchip using standard Illumina protocol.
Scan protocol
Arrays were imaged using BeadArray Reader (Illumina HiSeq2000) using standard recommended Illumina scanner setting
Data processing
Raw IDATs were processed with R (v4.1.1) package ‘SeSAMe’ with noob background correction, non-linear dye bias correction, and non-detection masking. P-value threshold was set at 0.1. Probe masking was performed using the standard mask list in SeSAMe, including probes that overlap with SNPs and repeat elements.
