single cell RNA-seq of human PBMCs
PBMCs were thawed quickly at 37oC and into DMEM supplemented with 10% FBS. Cells were quickly spun down at 400 g, for 10 min. Cells were washed once with 1 x PBS supplemented with 0.04% BSA and finally re-suspended in 1 x PBS with 0.04% BSA. Viability was determined using trypan blue staining and measured on a Countess FLII. Briefly, 12,000 cells were loaded for capture onto the Chromium platform using the single cell 3' gene expression reagent kit (v3 or v3.1) (10x Genomics). Following capture and lysis via Chromium Chip G, cDNA was synthesized and amplified (12 cycles) as per manufacturer's protocol (10x Genomics, protocols CG000204; CG000315). Amplified cDNA and libraries were checked for quality on Agilent 4200 Tapestation, quantified by KAPA qPCR, and sequenced on an Illumina NovaSeq 6000 targeting 100,000 raw read pairs per cell.