GSM8178028: PPPE.227 pp; Homo sapiens; RNA-Seq
Blood was diluted with PBS-10%FBS in a 1:1 ratio and added to Sepmate-50 tubes (Stemcell) containing 15mL Lymphoprep density gradient medium (StemCell). Tubes were centrifuged at 1200g for 10 minutes. PBMCs were collected and diluted with PBS for a final volume of 45mL and centrifuged at 300g for 10 mintues. Supernantants were removed and the pellet was resuspended with 10mL of PBS. A 10µl aliquot was taken and combined with 10µl trypan blue solution (Bio-Rad) for cell count on a Bright-Line hemacytometer (Hausser Scientific). The resuspended pellet was centrifuged at 400g for 10 minutes and the supernatant was discarded. Pellets were resuspended with FBS-10%DMSO at a final concentration of 2M cells/mL and aliquoted to be frozen down slowly at -80°C for 24-48 hours and then transferred to liquid nitrogen for long term storage. Library was constructed according to manufacturer instructions (10X Chromium Next GEM Single Cell 3' Reagent Kits v3.1 (dual index) with feature barcode technology) for cell multiplexing at the McGill Genome Cente