title: GSM2910020: Uninjuired Skin Hic1:tdTomato+ve; Mus musculus; RNA-Seq
library_construction_protocol: Large and small wounds from adult mice (n = 2 per sample) were dissected and enzymatically dissociated with dispase (StemCell Technologies) for 20 mins at 37 oC to remove the epidermis. Remaining dermis was dissociated using 0.2% collagenase for 2 hours at 37 oC. Cell suspensions were diluted with cold Hank's Buffered Salt Solution (HBSS; Life Technologies), strained through 40 µm cell filters (Falcon) and centrifuged at 280 X g. Liberated single cells were re-suspended in 0.5% bovine serum album in HBSS before FACS purification. For FACS purification, a stringent initial forward and side scatter gates were used to exclude debris, and cell doublets. Wildtype cells were used as negative control to determine gates for detection of tdTomato-fluorescence, then cells from Hic1CreERT2:tdTomato mice were purified. Libraries were prepared according to 10X Genomics ChromiumTM Single Cell 3' Reagent Guidelines v2 Chemistry as per the manufacturer's protocol. In brief, single cells were sorted into 0.1% BSA–PBS and partitioned into Gel Bead-In-EMulsions (GEMs) using 10xTM GemCodeTM Technology. This process lysed cells and enabled barcoded reverse transcription of RNA, generating full-length cDNA from poly-adenylated mRNA. DynaBeads® MyOneTM Silane magnetic beads were used to remove leftover biochemical reagents, then cDNA was amplified by PCR over 10 cycles. Quality control size gating was used to select cDNA amplicon size prior to library construction. Read 1 primer sequences were added to cDNA during GEM incubation. P5 primers, P7 primers, i7 sample index, and Read 2 primer sequences were added during library construction. Quality control and cDNA quantification was performed using Agilent High Sensitivity DNA Kit. Sequencing was performed first using Illumina MiSeq SR50 to approximate the number of recovered cells in each sample. We recovered 4061, 3776, 2604, and 3293 cells for Samples 1-4, respectively, with an estimated doublet rate of ≈3%. Based on this, we determined lane distributions for sequencing using Illumina HiSeq 4000 PE (75 bp paired-end reads) with a targeted sequencing depth of ~115,000 reads/cell.