title: GSM8296455: DTX, scRNAseq; Mus musculus; RNA-Seq
library_construction_protocol: Mouse wound bed and back skin were captured by 10mm punch, minced with scissors, and digested with 125 µg/ml Liberase TL (Roche, Basel, Switzerland) in RPMI. Minced samples were incubated at 37C for 1 hour 20 minutes with rotation, filtered through 70-µm and 40-µm filters, spun down, and resuspended in 2% FBS / PBS. 7AAD (BD Pharmingen, CA, USA) was added immediately before sorting. Live cells (7AAD-negative) sorting was performed on BD FACSAria III with FACS DIVA software (BD Biosciences, CA, USA). Single-cell capturing and library generation were performed using Chromium Single Cell 3' Reagents Kits v3 (10x Genomics, CA, USA) as the manufacturer's protocol. The sorted cells were resuspended at a concentration of approximately 1,000 cells/µL in PBS containing 0.04% BSA, and we targeted 10,000 cells per sample for capture. A single cell suspension was mixed with RT-PCR master mix and loaded with Single Cell 3' v3 Gel Beads and Partitioning Oil onto Chromium Chip. The chip was then loaded onto a Chromium Controller for single-cell GEM generation. Immediately following GEM generation, the Gel Bead is dissolved, primers are released, and any co-partitioned cell is lysed. Incubation of the GEMs produces barcoded, full-length cDNA from poly-adenylated mRNA. After GEM disruption, full-length cDNA is amplified to generate sufficient mass library construction. Finally, amplified cDNAs were fragmented, and adapter and sample index were added into finished libraries.