title: RNA-Seq of Mus musculus: hair follicle epithelium
design: Mouse adult dorsal telogen keratinocytes were isolated and stained for cell surface markers as described previously (Fujiwara et al., 2011) with some modifications. Lgr6-GFP-ires-CreERT2, Gli1-eGFP, and Cdh3-eGFP mice were utilized to fluorescently visualize the epidermal stem cells in the lower isthmus (Lgr6+), upper bulge (Gli1+), and hair germ (Cdh3+). Dissected dorsal skin of 8-week old mice was treated with 0.25% trypsin solution (Nacalai tesque) at 37°C for 1 hr, after which epidermal tissue was scraped off the dermal tissue. The separated epidermis was minced with scalpels and singularized via repeated pipetting. The cell suspension was stained with PE-Cy7-conjugated antibodies for CD45 (30-F11; eBioscience), TER-119 (TER-119; eBioscience), and CD31 (390; eBioscience) to identify haematopoietic and endothelial cells (lineage positive cells), as well as with Sca-1-PerCP-Cy5.5 (D7; eBioscience), CD34-eFluor660 (RAM34; eBioscience), CD49f (Integrin alpha6)-PE (GoH3; eBioscience). Cells were sorted with a FACSAria II after gating out dead and lineage positive cells. Hair germ epidermal cells (Cdh3+) were also sorted from the remaining dermal tissue in the same manner as in the epidermal tissue after incubation with 2 mg/ml of collagenase type I (Gibco) at 37°C for 2 hr with gentle mixing. Cells were subjected to RNA isolation with RNeasy Micro kit (Qiagen). Each library was prepared with 10ng of total RNA following the TruSeq Stranded mRNA Sample Prep Kit (Illumina) protocol, and sequenced using the Rapid Run mode with 80 cycles on the HiSeq1500 (Illumina).