Isolation of Murine Epidermal Stem Cells

Female C57BL6 mice were obtained from Janvier. EpSCs were isolated from telogen back skin collected from P56-60 mice, as previously described [27 (link)] with the following changes: fat and muscle tissue were removed from back skin using a scalpel. The skin was incubated in 0.5% Trypsin-EDTA (10X; Gibco,15400054) for 25 min at 37 °C on an orbital shaker. A single-cell suspension was then obtained by scraping the skin with a scalpel followed by neutralizing the trypsin by adding 1X PBS buffer containing 2% chelexed FBS (PBS-FBS(-)) (Gibco; 10010-015). The resulting cell suspension was then filtered through 70 µm and 40 μm cell strainers (Corning; 431750, 431751) and spun down. EpSCs were isolated using magnetic-associated cell sorting using Anti-SCA-1 microbeads (Miltenyi Biotec, 130-106-641) and a MACS MultiStand system (Miltenyi Biotec) together with MS columns (Miltenyi Biotec, 130-042-201). The resulting SCA-1+ EpSCs were spun down and resuspended in Trizol LS (Thermo Fisher, 102960-10). RNA was isolated using the Direct-zol™ RNA MiniPrep kit (Zymo Research, R2050) and concentration was determined using the Qubit^TM RNA BR assay kit (Invitrogen, Q10210). All animal procedures were approved by the Veterinary Office of the Canton of Zurich, Switzerland (License ZH233/2019).