single cell RNA-seq of human PBMCs
Peripheral blood mononuclear cells (PBMCs) were isolated by Lymphoprep (StemCell Technologies) gradient centrifugation within 1h after collection of blood samples by Lymphoprep (StemCell Technologies) gradient centrifugation whereas serum was isolated from the blood collected in red top collection tubes (BD Vacutainer). The isolated PBMCs were resuspended in a cryoprotective solution containing 90% FBS and 10% DMSO, then transferred to liquid nitrogen for long-term storage whereas serum was stored at -80°C. For scRNAseq, cells were washed and resuspended in PBS containing 0.04% BSA and immediately processed as follows. Cell viability was assessed on a Countess FL II automated cell counter (Thermofisher) using Trypan Blue staining, and up to 12,000 cells from each suspension were loaded onto one lane of a 10x Genomics Chip G. Single cell capture, barcoding, and library preparation were performed using the 10x Genomics Chromium Controller platform version 3.1 NEXTGEM chemistry and according to the manufacturer's protocol (CG000315, 10x Genomics). cDNA and libraries were assessed for quality via Tapestation 4200 (Agilent) and Qubit fluorometer (ThermoFisher), quantified by KAPA qPCR, and sequenced on a NovaSeq 6000 (Illumina) S4 v1.5 200 flow cell using a 28-10-10-90 asymmetric read configuration targeting a read depth of 50,000 per cell.