title: GSM8824012: PS mice, 8-weeks old, ATAC, pool of 4 mice; Mus musculus; OTHER
library_construction_protocol: Cell suspensions from adult mouse skin were obtained through mechanical dissociation and enzymatic digestion. Briefly, adult back skin from W8 or W24 mice was harvested in pre-digestion medium, finely minced and incubated at 37°C on a rotating plate to remove epithelial cells and other impurities. The samples were then digested for 45 minutes on a rotating plate with 1.25 mg of Liberase TM (Sigma) and 2.5 mg of DNAse I (Sigma) to disaggregate the tissue. Samples were further dissociated with the Miltenyi gentleMACS Dissociator and cells were then enriched with a Percoll gradient. Single cell suspensions were then magnetically sorted for CD45+ immune cells using Miltenyi CD45 microbeads. Single nuclei were isolated following the 10X Genomics Nuclei Isolation for Single Cell Multiome ATAC + Gene Expression Sequencing protocol (CG000365-Rev C). Briefly, approximately 150,000 single cells were lysed in chilled 0.025% IGEPAL Lysis Buffer (10X Genomics). The lysate was incubated on ice for 2 minutes, after which chilled Wash Buffer (10X Genomics) was added to quench lysis. The nuclei were then washed three times using Wash Buffer to remove cellular debris and residual lysis buffer components. Following the washes, the nuclei suspension was resuspended in Diluted Nuclei Buffer (10X Genomics) and filtered through a 40 μm strainer to remove aggregates and debris. Nuclei quality and concentration were assessed using Trypan Blue staining and quantified with a Countess 3 Automated Cell Counter (Thermo Fisher Scientific). Only preparations with greater than 80% intact nuclei and minimal debris were selected for downstream processing to ensure optimal sample integrity and sequencing efficiency. Nuclei were immediately treated using the 10X Genomics Chromium Next GEM Single Cell Multiome ATAC + Gene Expression protocol (CG000338-Rev F). Nuclei were diluted to the optimal concentration (9000–10 000 nuclei/μL) and loaded into the Chromium Next GEM Chip J, followed by partitioning into Gel Bead-in-Emulsions (GEMs) using the Chromium X (10X Genomics). Reverse transcription and ATAC transposition were performed simultaneously within individual GEMs according to the manufacturer's protocol. After GEM rupture, cDNA and transposed DNA fragments were purified using Dynabeads MyOne Silane (Thermo Fisher Scientific). Transposed DNA fragments underwent 7 PCR cycles amplification to generate chromatin accessibility libraries, while cDNA fragments were further processed through end-repair, adapter ligation, and 10 indexing PCR cycles to generate enrich gene expression libraries. The resulting gene expression and ATAC libraries were quantified using a Qubit Fluorometer (Thermofisher Scientific) and analyzed for fragment size distribution using an Agilent TapeStation before sequencing. The libraries were pooled at the recommended ratio and sequenced on an Illumina NovaSeq 6000 platform (Illumina) using paired-end reads (Read 1: 50 bp, Read 2: 49 bp, Index 1: 8 bp, Index 2: 24 bp), targeting a sequencing depth of ~25,000 read pairs per nucleus for ATAC ; and using paired-end reads (Read 1: 28 bp, Read 2: 90 bp, Index 1: 10 bp, Index 2: 10 bp), targeting a sequencing depth of ~50,000 read pairs per nucleus for gene expression to achieve a mean sequencing depth of 3350 median fragments per cell for a mean of 14946 cells post-aggregation.