title: GSM5467011: 4XE_ReindeerPBMC_Antler; Rangifer tarandus; RNA-Seq
library_construction_protocol: PBMCs were collected from venous blood supply from saphenous vein or from distal antler velvet into EDTA tubes and briefly stored on ice.  Blood was spun at 300g for 10 min and plasma 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.  Cells were washed twice with 5mL PBS, then spun at 300g 5 min.  Cells were resuspended in 2% BSA in PBS at a final concentration of 30,000 cells/100uL and run through a 40µm cell strainer, before loading into the 10X controller (~12,000 cells). 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.