GSM9131158: Keloid dermal cells, with AA, rep 4; Homo sapiens; RNA-Seq
Normal and keloid dermal fibroblasts (P8-9) were thawed and seeded in triplicate at a density of 26,300 cells/cm² (near confluency), and left overnight to adhere and spread. Cells were cultured in complete medium (high-glucose Dulbecco's Modified Eagle Medium (DMEM) with GlutaMAX and pyruvate (Gibco, #31966021) + 10% FBS and 1% penicillin/streptomycin (Sigma-Aldrich, #P0781)) with or without supplementation with 50μg/mL ascorbic acid for 5 days, with media changes every other day. After 5 days, triplicate wells were combined into 350μL RLT buffer (Qiagen, #74104), supplemented with 1% beta-mercaptoethanol (PanReac AppliChem, #A1108.0100) before being frozen at -80°C until downstream analysis. This experiment was repeated on serial passages, P8 & P9, which were used as technical replicates (N=4).   RNA was purified using the RNeasy Mini kit (Qiagen, #74104) following manufacturer's instructions, and quantified using a Qubit high sensitivity RNA assay (Invitrogen, #Q32852). RNA quality was ascertained by Bioanalyzer 2100 (Agilent). RNA Integrity Number (RIN) values above 9 were used. RNA from P8-9 normal and keloid fibroblasts cultured with and without ascorbic acid were shipped to BGI Genomics Co. Ltd., Hong Kong for library preparation and sequencing using the BGISEQ platform.