GSM5453488: HC20, BCR-Seq; Homo sapiens; OTHER
Retrieving the PBMC-containing cryo-tubes from the -196℃ liquid nitrogen storage tank and placing them in a 37℃ water bath for rapid thawing. PBMCs were mixed with 10 mL washing medium (90% DMEM+10% FBS) in a 15-mL polypropylene tube and centrifuged at 500g for 20 min. The supernatant was then aspirated (repeat twice). The cell pellets were resuspended with 500 μl× PBS (0.04% BSA) in the sterile RNase-free vacutainer tubes and added with 5 ml 1× red blood cell lysis buffer (MACS 130-094-183, 10×) and incubated at room temperature for 10 min to lyse remaining red blood cells. After incubation, the suspension was centrifuged at 500g for 20 min at room temperature. The suspension was resuspended in 100 μl Dead Cell Removal MicroBeads (MACS 130-090-101) and remove dead cells using Miltenyi ® Dead Cell Removal Kit (MACS 130-090-101). Then the suspension was resuspended in 1× PBS (0.04% BSA) and centrifuged at 300 g for 3 min at 4 °C (repeat twice). The cell pellet was resuspended in 50 μl of 1× PBS (0.04% BSA). The overall cell viability was confirmed by trypan blue exclusion, which needed to be above 85%, single cell suspensions were counted using a Countess II Automated Cell Counter and concentration adjusted to 700-1200 cells/μl. manufacturer's protocols for 5'barcoded GEX and V(D)J BCR