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 plasma was isolated from the blood collected in ACD collection tubes (BD Vacutainer) by centrifugation. 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 plasma 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 LUNA FL automated cell counter (LOGOS Biosystems) using AOPI staining, and up to 29,000 cells from each suspension were loaded onto one lane of a 10x Genomics Chromium GEM-X Single Cell 5' Chip v3. Single cell capture, barcoding and library preparation were performed using the 10x Genomics Chromium X platform 5' version 3 GEM-X chemistry and according to the manufacturer's protocol (CG000733, revA). From each individual sample, cDNA was derived and used for the construction of a gene expression and a TCR (VDJ) library. cDNA and libraries were checked for quality by Tapestation 4200 (Agilent) and Qubit Fluorometer (ThermoFisher) and quantified by KAPA qPCR. Libraries were sequenced on an Illumina NovaSeq X+ 25B 300 cycle flow cell, with a 28-10-10-90 (R1-I1-I2-R2) asymmetric read configuration, targeting 20,000 barcoded cells with an average sequencing depth of 50,000 reads per cell and 15,000 reads per cell for the gene expression and TCR library.