title: GSM7025079: Control, replicate 1, scRNAseq; Mus musculus; RNA-Seq
library_construction_protocol: Sterile PBS on ice was used to preserve skin tissue samples immediately after dissection. The samples were subsequently cleaned by PBS twice and cut into pieces sized 2-4 mm. Then tissues were processed following the instructions of the dissociation kit (Multi Tissue Dissociation Kit 1, Miltenyi Biotec, 130-110-201) to complete the dissociation. The single-cell suspension was passed through 70 and 40 μm cell strainers and centrifuged for 10 min, 500 × g at 4 °C. The red blood cell (RBC) lysis solution (130-094-183, Miltenyi Biotec) and Dead Cell Removal Kit (130-090-10, MiltenyiBiotec)were successively added to remove erythrocytes or dead cells. The process resulted in highly viable, typically ≥85%, single-cell suspensions. For immediate single-cell capture, the cells were resuspended in 0.04% Ultra-Pure BSA in PBS (Thermo Fisher Scientific) and concentration was adjusted to ≥106 cells/ml. The utilization of microdroplets in conjunction with barcoded primer beads enabled the capture of individual cells through the implementation of a droplet-based ultra-high throughput system for parallel gene expression detection. The Chromium Next GEM Single Cell 3ʹ Kit v3.1, developed by 10x Genomics (Part Number 1000268), was employed in the creation of gene expression (GEX) libraries. The generation of gel bead-in-emulsions, as well as the reverse transcription (RT) of the single-cell suspensions, was accomplished through the utilization of the 10x Genomics Single cell chip (Chromium Next GEM Chip G Single Cell Kit, Part Number 1000120, and an additional Dual Index Kit TT Set A, Part Number 1000215) run on the Chromium Controller (Part Number 110211) developed by 10x Genomics. The generated cDNA was amplified in order to produce GEX libraries after the RT step, subsequently quantified through the use of Qubit 3.0 fluorometer (Life Technologies, Part Number 15387293) and assessed through the utilization of HS DNA chips (Agilent Technologies, Part Number 5067-4627) in combination with the 2100 Bioanalyzer (Agilent Technologies, Part Number G2939BA). The Novaseq 6000, developed by Illumina, was employed for massively parallel sequencing.