biostudies-arrayexpressRaha TaghaviHomo sapiensIllumina Command Software v1.3.159007cellranger-9.0.0https://www.ebi.ac.uk/biostudies/studies/E-MTAB-15435scRNA-seq was performed to characterise an in vitro model of T cell exhaustion. CD8+ T cells from two donors underwent chronic stimulation to induce exhaustion and samples were collected from multiple points across the protocol to examine the subsets and transcriptional signatures present.biostudies-arrayexpressSample Collection - 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).Sequencing - The library of 16 samples was sequenced in 100bp paired-end read configuration across five lanes of an Illumina NovaSeq X plus 10B flowcell, to a total depth of 2362M Paired-end reads.Sample Treatment - 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).Growth Protocol - 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.Library Construction - 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).Nucleic Acid Extraction - 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)OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesData Transformation - Sequencing data was processed using Cell Ranger software (cellranger-9.0.0; cellranger-multi pipeline) to align reads to the Chromium Human Transcriptome Probe Set v1.0 and demultiplex samples.MetabolomicsUnknownTranscriptomicsGenomicsProteomicsIllumina NovaSeq XRNA-seq of coding RNA from single cellsHomo sapiensReversing T cell dysfunction in a novel in vitro model of T cell exhaustion reveals differential roles of RASA2Rachael NicholsonRaha TaghaviRebecca GrahamSofia LourencoHilal Sarac, Rachael Nicholson, Rebecca N. Graham, Meera Augustus, Raha Taghavi, Dympna J. Connolly, Lindsay Lim, Sofia LourencofalsescRNA-seq characterisation of in vitro-generated CD8+ exhausted human T cellsscRNA-seq was performed to characterise an in vitro model of T cell exhaustion. CD8+ T cells from two donors underwent chronic stimulation to induce exhaustion and samples were collected from multiple points across the protocol to examine the subsets and transcriptional signatures present.2026-05-31T00:00:00Z2026-05-31T01:01:14.057Z2025-08-01T13:07:04.833ZE-MTAB-15435ERP177709EFO_0002944EFO_0004170EFO_0003789EFO_0005684EFO_0005518EFO_0003816EFO_0004184EFO_0003969