For FACS sorting of HFSCs and BL cells, the protocol has been described previously (Lee et al., 2016 (link)). The epithelial cells were isolated from the back skin through trypsin digestion. The cell suspensions were labeled with anti-CD34-Biotin (1:50, no. 13-0341-85, eBioscience), α-Streptavidin-APC (1:100, no. 554067, BD Biosciences) and anti-α6-integrin-PE (1:40, CD49f, no. 555736, BD Biosciences) to isolate HFSCs (CD34+, α6-integrin+) and BL cells (CD34−, α6-integrin+). Propidium iodide (1:1,250–1:2,500 of 1 mg mL−1 stock, S7109, Sigma) was used to rule out the dead cells. For negative controls to gate the fluorescence-labeled cells, we used only α-Streptavidin-APC for CD34+ cells and PE-Rat-IgG2a (1:40, no. 555844, BD Biosciences) for α6-integrin+ cells. The cells were isolated with BD FACSAria located in the Flow Cytometry Core at Cornell University.
For gene expression analysis, RNA was extracted from either 1 cm2 of total skin using the RNeasy Fibrous Tissue Mini Kit (no. 74704, QIAGEN) or the FACS-sorted cells using the mirVana miRNA Isolation Kit (AM1591, Ambion). The cDNA were synthesized using iScript (no. 1708841, Bio-Rad) cDNA synthesis kit and qRT-PCR analysis was described previously (Lee et al., 2016 (link)).
For gene expression analysis, RNA was extracted from either 1 cm2 of total skin using the RNeasy Fibrous Tissue Mini Kit (no. 74704, QIAGEN) or the FACS-sorted cells using the mirVana miRNA Isolation Kit (AM1591, Ambion). The cDNA were synthesized using iScript (no. 1708841, Bio-Rad) cDNA synthesis kit and qRT-PCR analysis was described previously (Lee et al., 2016 (link)).