title: GSM8991934: KOD3-2; Mus musculus; RNA-Seq
library_construction_protocol: Wound tissues were collected from male and female Mfap5+/+ or Mfap5-/- mice (n=4-5 animals for each sex and each experimental group) on days 0 (D0), 3 (D3), and 7 (D7) post-wounding. A total of 48-60 mice were used for single-cell RNA-sequencing. Collected wound tissues were pooled separately based on sex, experimental group, and time post-wounding. Tissue collected from D0 was considered NS. After collection and pooling, tissues were micro-dissected, minced, and incubated at 37°C in a dispase (Worthington Biochemical Corporation, Lakewood, NJ, Catalog # 9001-92-7)/liberase (Roche, Basel, Switzerland, Catalog # 5401135001) solution supplemented with DNAse I (Sigma-Aldrich, Catalog # D5025) and penicillin-streptomycin (Gibco, Waltham, MA, Catalog # 15-140-122) for 60 min with constant rotation. The final concentrations for each solution component were 3.6 mg/mL dispase, 30 µg/mL liberase, 100 µg/mL DNAse I, and 100 Units/mL-100 µg/mL penicillin-streptomycin. Post-incubation, cell suspensions were passed through a 40 µM cell strainer (ThermoFisher Scientific, Waltham, MA). Single-cell suspensions were treated with 1x RBC lysis buffer (Biolegend, San Diego, California, Catalog # 420301), washed, and resuspended in cold PBS before debris removal using the Debris Removal Solution (Miltenyi Biotec, North Rhine-Westphalia, Germany, Catalog # 130-109-398) according to the manufacturer's protocol. All cells were then frozen in a mixture of 70% DMEM (Corning Inc, Corning, NY), 30% Fetal Bovine Serum (FBS) (GeminiBio, Sacremento, CA), and 10% DMSO (Sigma-Aldrich) before being sent to Novogene (Novogene America, Sacramento, CA). Thawed cell suspensions underwent dead cell removal, and only samples with >100,000 cells, >70% viability, and minimal to no cell clumps were used for single-cell RNA sequencing. GEM generation, barcoding, post-GEM-RT cleanup, cDNA amplification, and cDNA library construction were performed using Single-cell 3′ v3 chemistry (10X Genomics, Pleasanton, California). Libraries were then sequenced on an Illumina NovaSeq 6000 (Illumina, San Diego, CA). Dead cell removal, cell counting and viability testing, GEM generation, barcoding, post-GEM-RT cleanup, cDNA amplification, library preparation, quality control, and sequencing were all performed by Novogene (Novogene America). Transcripts were mapped to the mouse reference genome (GRCm38/mm10) using Cell Ranger Version 7.0.0. cDNA library construction was performed using Single-cell 3′ v3 chemistry (10X Genomics, Pleasanton, California). scRNA-seq