title: GSM9161708: skin cells,DTA,M927; Mus musculus; RNA-Seq
library_construction_protocol: Mice were anesthetized, and the dorsal hair was shaved and removed using a depilatory cream. Dorsal skin tissues were excised, and subcutaneous fat was carefully scraped using a surgical scalpel. The harvested tissues were immediately transferred to a culture dish containing ice-cold 1× phosphate-buffered saline (PBS) devoid of RNase, calcium, and magnesium ions. Tissues were then cut into ~0.5 mm² fragments and washed repeatedly with 1× PBS to eliminate surface blood, oil, and other contaminants。The tissue fragments were incubated in a dissociation solution comprising 0.35% collagenase IV, 2 mg/mL papain, and 120 U/mL DNase I at 37°C for 20 minutes in a shaking water bath (100 rpm). Tissue digestion was terminated by adding 1× PBS supplemented with 10% fetal bovine serum (FBS), followed by gentle pipetting (5–10 times) to further dissociate cells while minimizing shear-induced damage. The resulting suspension was filtered sequentially through 70 μm and 30 μm cell strainers, then centrifuged at 300 × g for 5 minutes at 4°C. The cell pellet was resuspended in 100 μL of 1× PBS containing 0.04% bovine serum albumin (BSA). To remove red blood cells, 1 mL of 10× red blood cell lysis buffer (MACS, Cat# 130-094-183) was added, and the sample was incubated for 2–10 minutes at room temperature or on wet ice. After lysis, the suspension was centrifuged again at 300 × g for 5 minutes. The pellet was resuspended in 100 μL of 1× PBS (0.04% BSA), and dead cells were removed using Dead Cell Removal MicroBeads (MACS, Cat# 130-090-101) following the manufacturer's instructions. The procedure was repeated twice, each with centrifugation at 300 × g for 3 minutes at 4°C. The final single-cell suspension was prepared by resuspending the cell pellet in 50 μL of 1× PBS (0.04% BSA). Cell viability was assessed using trypan blue staining, with samples required to exceed 85% viability. Cell counts were determined using a hemocytometer, and cell concentrations were adjusted to 700–1200 cells/μL. Single-cell suspensions were processed using the 10x Genomics Chromium system to capture approximately 5,000 individual cells, following the manufacturer's instructions as detailed in the Chromium Single Cell 3' Reagent Kits User Guide (v3). Subsequent cDNA amplification and library preparation were performed according to the standard protocol. The resulting libraries were sequenced on an Illumina NovaSeq 6000 platform (paired-end, 150 bp) by LC-Bio Technology Co., Ltd. (Hangzhou, China), achieving a minimum sequencing depth of 20,000 reads per cell.