scRNA-seq of diabetic mouse 10 day wounds: adult male skin
Skin cells were pooled from four wounds (two wounds from each of two mice per genotype). After washing, red blood cells were lysed and removed from the single cell suspension using 1x Red Blood Cell Lysis Solution (Miltenyi Biotec, Gaithersburg, MD, USA), followed by debris removal with Debris Removal Solution (Miltenyi Biotec) according to the manufacturers instructions. Cells were then resuspended in cold 1x DPBS and passed through a 30 m Pre-Separation Filter (Miltenyi Biotec). After centrifugation, dead cells were removed using the Dead Cell Removal Kit (Miltenyi Biotec) following the manufacturers instructions. Briefly, cells were incubated with an optimized volume of Dead Cell Removal MicroBeads at room temperature for 15 min. Subsequently, 1x Binding Buffer was added to a final volume of 500 L, and cells were passed through an MS MACS column system (Miltenyi Biotec) for a negative magnetic selection, with a total of four separations. Final live cells were washed, counted, and resuspended in cell staining buffer (BioLegend), and processed for surface protein labeling. Next, cells were incubated with TruStain FcX PLUS (anti-mouse CD16/32) antibody (BioLegend) on ice for 10 min to block Fc receptors, followed by staining with the TotalSeq-A antibody pool (BioLegend) on ice for 30 min according to the manufacturers instructions. A complete list of antibodies is provided in Table 1. Cells were then washed three times with cell staining buffer, filtered through a Flowmi Cell Strainer (40m, SP Bel-Art, Wayne, NJ, USA), and resuspended in 1x DPBS supplemented with 10% fetal bovine serum. After cell counting and viability assessment (>85% live cells), samples were immediately processed using the Chromium platform (10x Genomics, San Francisco, CA, USA). Gene and surface protein libraries were constructed according to the Chromium GEM-X Single Cell 3 Reagent Kits v4 User Guide (CG000732, 10x Genomics) and the TotalSeq-A antibodies with 10x Single Cell 3 Reagent Kit v3 protocol (BioLegend), and sequenced on a NovaSeq 6000 Sequencing System (Illumina, San Diego, CA, USA).