GSM8560028: Control Skin IgG 3; Mus musculus; OTHER
Depilated Control and PITX1+ mice dorsal skin (n=3 separate mice for each condition) was incubated in 2U/mL of dispase II for 2 hours at 25°C with gentle rotation. Skins were rinsed twice in 1X PBS before briefly drying and spreading dermis-side down on a 10mm petri dish. A #10 scalpel blade held perpendicularly to the skin was used to gently scrape the epidermis off and pool it into a gentleMACS C-Tube (Miltenyi). The Whole Skin Dissociation Kit (Miltenyi) was then used to dissociate the tissue per manufacturer's protocol. Tissues were incubated in enzymatic mixture for 1 hour at 37°C prior to cell dissociation: one cycle of 'h_skin_01' on gentleMACS Dissociator (Miltenyi), filtration of cells through 70µm and 30µm Preseparation Filters (Miltenyi), and resuspension and counting in KBM-Gold (Lonza). The vast majority (>95%) of cells were keratinocytes (intended cell type to collect) with the ~5% remaining cells consisting of melanocytes, epidermal-resident immune cells, and stray fibroblasts. 100,000 single epidermal cells were processed with the CUTANA CUT&Tag Kit v1 (Epicypher) for nuclear isolation, chromatin tagmentation, and library preparation. Cells were gently fixed in 0.1% methanol-free formaldehyde for 1 minute at room temperature and neutralized in 125mM glycine prior to nuclear extraction. 16 PCR cycles were used to amplify tagmented libraries prior to sequencing.