title: GSM8568223: WT_RTX_72H_1; Mus musculus; RNA-Seq
library_construction_protocol: Cells were isolated from C57BL/6J mice in first anagen after 12 hr injection with saline (control) or RTX, and after 72 hr injection with RTX. Mouse back skin containing the damaged regions was dissected, and the fat layer was removed by scraping with a surgical scalpel. The dermal fraction was isolated by incubating the skin in a collagenase solution in HBSS on an orbital shaker for 45-60 minutes at 37°C with scraping of the dermis. Following collection of the dermis, the remaining skin was incubated in trypsin-EDTA for 25-30 minutes at 37°C to collect the epidermis. Single cell suspensions of each fraction were obtained by filtering through a 70-mm and 40-mm filter. Cell suspensions were then centrifuged for 8 minutes at 350g at 4°C and resuspended in 5% FBS with a CD16/F32 Fc blocking antibody (BioLegend 101302, 1:100). Libraries were constructed according to the 10x Genomics Single Cell v3.1 protocol For scRNAseq, a small aliquot of the epidermal and dermal fractions was set aside containing fibroblasts. The fibroblasts were depleted from the remainder of each fraction by incubating with CD140A-biotin antibody (eBioscience 13-1401-82, 1:200) and then removing CD140A-bound cells with magnetic beads. The aliquoted fractions were then added back to respective samples, and cells were stained for 45-60 minutes on ice using the following antibodies: CD34-e660 (eBioscience 0-0341-82, 1:100), Ly-6A/E (Sca-1)-PerCp-Cy5.5 (eBioscience 45-5981-82, 1:1000), CD45-eF450 (eBioscience 48-0451-82, 1:250), a6-PE (eBioscience 12-0495-82, 1:250). DAPI (1:1000) was used to exclude dead cells. FACS was used to enrich for cell populations of interest at a ratio of 2:1:3:1:2 (immune cells: fibroblasts: hair follicle: general cells: HFSCs) that were isolated as follows: immune cells (CD45+), fibroblasts (Sca1+, CD34+, a6-), hair follicle (Sca1-, CD34-,a6low-high), general cells (DAPI-, CD45-, Sca1-, CD34-,a6-), HFSCs (Sca1-, CD34+,a6+).