title: GSM8666265: RNA from TSP2KO mouse (3mos) dermal fibroblasts sample2; Mus musculus; RNA-Seq
library_construction_protocol: Dermal fibroblasts (DFs) were isolated from 12-week-old WT and TSP2KO mouse dorsal skin according to the established protocol. Briefly, mouse dorsal skins were shaved, excised, treated with 5% antibiotic-antimycotic (Gibco), and incubated in 25 μg/ml Trypsin (Sigma) overnight at 4°C. After separation from the epidermis and adipose tissue, the dermis was physically disrupted and then digested using collagenase IV (Worthington Biochemical) for 3 hrs under 37 °C. After centrifuge, cell pellet was then resuspended in 25mM glucose Dulbecco's Modified Eagle Medium (DMEM, Gibco) with 10% (v/v) fetal bovine serum (FBS, Peak Serum Inc) and 1% (v/v) penicillin-streptomycin (P/S, Gibco) and culture for 3-5 days to enrich the fibroblast population. For single-cell RNA sample preparation, after the enrichment assay, fibroblasts were lifted using trypsin (Sigma), suspended in PBS at a concentration of 1000 cells/µl, and passed through a 100 µm cell strainer (BD Bioscience). For bulk RNA sequencing, RNA was isolated from passage 1 fibroblasts using the RNeasy Mini Kit (Qiagen) according to the manufacturer's protocol. The single-cell suspension was sent to the Yale Microarray Center, where library preparation was performed using the Chromium Next GEM single-cell 3' v3 reagent kit (10x Genomics). For bulk-RNA sequencing, approximately 2 µg of RNA per sample was sent to the Yale Genomics Core for sequencing using the Illumina TruSeq Stranded mRNA kit (Illumina).