title: GSM8072974: EpiKO_ZT20_1; Mus musculus; RNA-Seq
library_construction_protocol: Mice were sacrificed at the relevant time by C02 asphyxiation, and their backskin was subsequently removed and stored in ice-cold PBS. The hypodermis was then removed from the skin by vigorous scraping using a scalpel. In a petri dish, the skin was floated on 10ml of 1mg/ml dispase (Sigma Aldrich) (in PBS) for 45 minutes at 37°C to enzymatically separate the epidermis. Using a scalpel, the epidermis was separated from the surface of the skin, and then mechanically disaggregated using surgical scissors. The disaggregated epidermis was placed into 10ml of ice cold PBS + 10% chelated FBS to inactivate dispase, and then sequentially filtered through 100µM and 40µM filters, giving a purified extract of epidermal cells. To extract total RNA, one million epidermal cells were lysed in 500µL TRIzol (Invitrogen), vortexed with 100µL chloroform, and then the resulting aqueous layer containing RNA was isolated. The aqueous layer was mixed with 825µL RLT buffer (Qiagen) and 625µL 100% ethanol to induce RNA precipitation. The sample was further processed using an RNeasy mini kit (Qiagen) in accordance with the manufacturers protocol, and RNA was eluted in molecular biology grade water. Prior to library preparation, RNA integrity was estimated using an RNA 6000 Nano Bioanalyzer 2100 Assay (Agilent). Libraries were prepared with KAPA Stranded mRNA-Seq Illumina® Platforms Kit (Roche) following the manufacturer´s recommendations. The libraries were sequenced on NovaSeq 6000 (Illumina) with a read length of 1x51bp+17bp+8bp using, following the manufacturer's protocol for dual indexing. Image analysis, base calling and quality scoring of the run were processed using the manufacturer's software Real Time Analysis (RTA 3.4.4).