subexperiment: TERT immortalization coriell_id: AG06561 cell_line: Fetal_skin_fibroblast_1 donor_age: 16 fetal weeks donor_sex: female culture_oxygen: Ambient culture_agent: NA culture_pctfbs: Full expression_vector: TERT subculture: 2b population_doublings: 122.74 days_in_culture: 227 batch_date: 09.24.21 growth protocol: Fibroblasts were maintained in Eagle’s MEM with Earle’s salts and non-essential amino acids with 15% v/v fetal bovine serum. 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: Low-PD primary fibroblasts were transduced with purified lentiviral particles containing expression vectors encoding human Telomerase Reverse Transcriptase (TERT) and hygromycin resistance marker. Following selection with 250ug/ml hygromycin B, cells were serially cultured in perpetuity, in triplicate.
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.
