GSM7438945
For mice, dorsal skin was isolated, subcutaneous fat was removed, and was cut into small pieces prior to digestion with 0.25% collagenase type IV in Dulbecco's Modified Eagle Medium at 37C for 1 hour in a shaking incubator. Collagenase was neutralized with Advanced RPMI 1640 Medium + 10% FBS and the digested skin was passed through a 70um strainer. The flow through was rinsed twice with centrifugation at 350g, 4C. Single-cell suspensions were stained with anti-CD45-FITC antibody and propidium iodide. CD45+, propidium iodide- cells were isolated via FACS. For human samples, human scalp biopsies were digested using the Whole Skin Dissociation Kit and its accompanying protocol, which involved the use of a gentleMACS Dissociator. Cells were resuspended and washed in 2% FBS in Cell Staining Buffer, then stained with DRAQ5 and SYTOX-Green prior to FACS isolation for DRAQ5- SYTOX-Green- live cells. Libraries were prepared using the 10x Genomics Chromium Single Cell 3' Reagent Kits (version 2 for mouse, version 3 for human samples) according to the manufacturer's instructions, with a target of 5000 cells and 350 million reads