GSM6268364: Reindeer_coculture_antler_velvet_R_CA; Rangifer tarandus; RNA-Seq
Whole blood were collected 3 days post-wound from venous blood supply into EDTA tubes. Blood was spun at 300g for 10 mins and plasma was removed and stored at -80oC. Buffy coat was collected, and red blood cells were lysed with ACK buffer (1:10 ACK to blood cells) for 5 minutes and then spun at 300g for 5min and then supernatant. To liberate fibroblasts from reindeer back skin and velvet, either a 12mm circular biopsy punches (velvet) or an elliptical skin excision (back skin) was collected and dissociated to single cells by mincing the skin into 1–2 mm pieces using a pair of scalpels. Minced tissue was first submerged in dispase (2 mg/ml at 37°C for 10 minutes, Stem Cell Technologies) to remove epidermis and then in collagenase type IV (2 mg/ml at 37 for 1-3 hours with mechanical trituration at set intervals, Sigma) to digest the dermal extracellular matrix. Dissociated cell suspensions were passed through 100μm and then 70μm cell strainers, centrifuged at 1200rpm for 6 minutes to pellet the liberated cells, and resuspended in complete fibroblast culture media (DMEM + 10%FBS, 5mL of L-glutamine, penicillin-streptomycin). Fibroblasts were expanded for three days in a 37 °C 5% CO2 tissue culture incubator prior to initiating co-culture with circulating immune cells. Following co-culture incubation, immune cells cells were resuspended in 2% BSA in PBS prior to loading into the 10X controller. The sample was prepared according to 10X Genomics ChromiumTM Single Cell 3' Reagent Guidelines v3 Chemistry. Briefly, single cells were 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.