title: GSM9253347: CTL skin wound 2, Day 4 following injury; Mus musculus; RNA-Seq
library_construction_protocol: One skin wound was processed per sample. Skin tissue was minced into small pieces, then digested with 10 ml of DMEM/F12 containing 1.5 mg/ml collagenase I, 10% FBS for 2 hours at 37°C with agitation. Samples were centrifuged, and the cell-containing supernatant was passed through a 40-μm cell strainer and set aside on ice. The remaining skin pieces were further digested with 2 ml DMEM/F12 containing 0.025% Trypsin-EDTA  and 2 mM EDTA for 5 minutes at 37°C with agitation. Trypsin was inactivated and the remaining skin pieces were passed three times through an 18G needle and five times through a 21G needle. The digested skin suspension was then filtered through a 40-μm cell strainer and combined with the previously obtained cell suspension that was kept on ice. Samples were washed once with HBSS, centrifuged, and resuspended in DMEM/F12 medium containing 10% FBS, 1% Penicillin-Streptomycin, and 1:1000 Protector RNase Inhibitor. Debris and cell clumps were filtered out manually using 40-μm mesh filters. Dead cells were then removed with EasySep™ Dead Cell Removal (Annexin V) Kit (STEMCELL Technologies, #17899), according to manufacturer's instructions. Single cell libraries were generated using the Chromium Next GEM Single Cell 3' GEM, Library & Gel Bead Kit v3.1 (10x Genomics). 7000 cells per sample were targeted for capture. Each library was sequenced on the Illumina NovaSeq 6000 platform to achieve approximately 50000 reads per cell.