title: GSM5696732: Ectopic_Velvet_Graft_scRNA_6week_post-graft_5XD; Rangifer tarandus; RNA-Seq
library_construction_protocol: Following the collection of ectopic skin excision, the sample was minced and incubated in dispase (StemCell Technologies) for 10-15 minutes at 37°C to remove epidermis. Remaining dermis tissue was minced and digested using collagenase (0.2%, Sigma) in 37 degree C water bath for 1-3 hours. To further dissociate the tissue and liberate single cells, the gentleMACS™ Octo Dissociator (Miltenyi Biotec) was used for 6 minutes on a homogenization program. DNase I (1x - Sigma) was also added to the tissue for the last 30 minutes in the water bath. Single cell suspensions were resuspended in cold FACS staining buffer (1% bovine serum albumin in Hank's Buffered Salt Solution, HBSS; Life Technologies), strained through 100 µm and 70 µm filters (Falcon) and centrifuged at 1200 rpm for 6 minutes. Cell pellets were resuspended in FACS staining buffer and quantified. Viability dye eFlour780 (eBiosciences) was used to exclude dead cells using FACSAria III (BD Biosciences). Of note, identical tissue processing steps were followed as described in GSE142854 to enable side-by-side comparisons with Day 0 Back and Day 0 Antler scRNA-Seq samples. The sample was prepared according to 10X Genomics ChromiumTM Single Cell 3' Reagent Guidelines v3 Chemistry. Briefly, single cells were sorted into 0.1% BSA–HBSS 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 using Illumina NovaSeq S2 flow cell at the Center for Health Genomics and Informatics at the University of Calgary.