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 System using the v2 single cell reagent kit (10X Genomics). Following capture and lysis, cDNA was synthesized and amplified (12 cycles) as per manufacturer's protocol (10X Genomics). The amplified cDNA was used to construct an Illumina sequencing library and sequenced on a single lane of a HiSeq 4000. FASTQ files for each sample (10X chemistry v2) were generated and demultiplexed using Illumina bcl2fastq 2.20.0.422.