scRNA-seq characterisation of in vitro-generated CD8+ exhausted human T cells
scRNA-seq characterisation of in vitro-generated CD8+ exhausted human T cells
PBMCs were isolated from leukocyte cones from healthy donors (NHSBT) using SepMate™ Isolation Tubes (StemCell Technologies, #85450) and subsequently CD8+ T cells were isolated using EasySep™ Human T cell isolation kits (StemCell Technologies, #17953). Cells were cultured in RPMI-1640 (Gibco Thermo Fischer Scientific, 21875091) media supplemented with 10% Heat Inactivated (HI) FBS and 50 IU/mL rhIL-2 (R&D Systems, #202-IL-050). Cultures were maintained at 37 °C in humidified incubators with 5 % CO2. In vitro, cells were stimulated with aCD3/aCD28 T Cell Activator (Stem Cell Technologies, #10971). Samples received either serial stimulations (Tex) or a single stimulation (Ts).  Cells were fixed and permeabilised at 4°C for 16 hours according to 10x Genomics guidelines (CG000782, Rev C). Gene expression was measured using barcoded probe pairs designed to hybridize to mRNA specifically. Using a microfluidic chip, the fixed and probe-hybridized single cell [or nuclei] suspensions were partitioned into nanolitre-scale Gel Beads-in-emulsion (GEMs). A pool of ~737,000 10x GEM Barcodes was sampled separately to index the contents of each partition. Inside the GEMs, cells were lysed, probes were ligated and the 10x GEM Barcode was added, and all ligated probes within a GEM share a common 10x GEM Barcode. Steps were performed as per 10x Genomics guidelines (User Guide: CG000787 GEM-X Flex Gene Expression Reagent Kits - Multiplex) Barcoded and ligated probes were then pre-amplified in bulk, after which gene expression libraries were generated as per 10x Genomics guidelines (User Guide: CG000787 GEM-X Flex Gene Expression Reagent Kits - Multiplex).