GSM6588787: 1_week_experimental; Mus musculus; RNA-Seq
DNp63fl/fl (control) and DNp63fl/fl;K14-CreT2 (experimental) mice, age 2-17 months, 50:50 males/females ratio, were treated with 200mg/kg Tamoxifen to genetically ablate DNp63 in the basal epidermal cells (basal keratiocytes). Primary mouse keratinocytes were isolated after 1 day or 1 week post-ablation as previously described (Jensen KB, Driskell RR, Watt FM. Nat Protoc. 2010;5(5):898-911) with slight modifications. Mice were euthanized by CO2 asphyxiation and the back hair removed using hair clippers and Nair hair remover lotion. The skin was washed with 70% ethanol, dissected, and placed the dermal side up on a 10 cm plate on ice, the muscle and adipose tissue was carefully removed with a cell scraper and forceps. The skin was then floated epidermal side up in 10 ml 0.25% trypsin without EDTA (Gibco) either overnight at 4°C or for two hours at 37°C, transferred to a new 10 cm plate, and the epidermal layer was carefully scraped off the dermis using a cell scraper. The epidermis was then minced with a razor blade and clumps were disaggregated by pipetting in trypsin. Ice-cold PBS/10% FBS was added to the cell suspension and passed through a 40 mm mesh. Isolated KCs were gently pelleted and washed twice with ice-cold PBS/10% FBS. Cell number and viability was determined using Trypan blue (Sigma) and the Beckman Coulter Z1 Counter (Beckman Coulter). At 1 day post-ablation, Chromium Next GEM Single Cell 3′ GEM, Library & Gel Bead Kit v3.1, PN-1000121 was used. At 1 week post-ablation, Chromium Next GEM Single Cell 5′ Library & Gel Bead Kit v1.1, PN-1000165 was used.