GSM9396079: pool-14, well-3, GEX; Homo sapiens; OTHER
Blood was collected in EDTA tubes within a 90 min window for all participants of the study. Peripheral blood mononuclear cells (PBMC) were obtained by density gradient centrifugation of whole blood at 800g for 30 min at room temperature without brake, using Ficoll-Paque (Cytiva). Isolated cells were cryopreserved in a solution containing fetal Bovine Serum and 10% DMSO (Sigma). Thawed PBMC suspensions from 14-16 individuals were pooled together with equivalent numbers to a total of 1 million live cells. Pools were resuspended in Cell Staining Buffer (Biolegend) and incubated with Fc Receptor Blocking Solution (Biolegend) at 20mg/mL for 10 minutes on ice. Cells were then stained with a cocktail of 140 TotalSeq-C oligo-tagged antibodies (Biolegend) for 30 minutes at 4°C. Stained cells were washed in PBS containing 1% BSA, filtered through a 70uM filter, and re-suspended in a final solution of PBS containing 0.04% BSA. Stained cells (n=60,000) were loaded on the Chromium Controller (10X Genomics) for GEM generation. The cDNA libraries were generated using the Chromium Next GEM Single Cell 5' v1.1 Library Kit for gene expression (GEX) and the Chromium Single Cell 5' Feature Barcode Library kit for antibody derived tag (ADT) according to manufacturer's instructions (10X Genomics). Each batch of 14-16 samples was sampled four times to prepare four independent cDNA libraries per batch to obtain a sufficient number of cells per sample. The libraries were subsequently sequenced on a NovaSeq 6000 S4 platform (Illumina).