mRNA sequencing dataset of early T cell activation. In short, in vitro cultured T cells were activated using anti-CD3 and anti-CD28 antibodies. Cells were harvested at 0, 2, 4, 6, and 24 hours post-activation, pelleted, and snap-frozen. Pellets were used for mRNA sequencing.
Peripheral blood mononuclear cells (PBMCs) from anonymized healthy donors were used in accordance with the Declaration of Helsinki (Seventh Revision, 2013) after written informed consent (Sanquin). PBMCs were isolated through Lymphoprep density gradient separation (Stemcell Technologies). For proteomic and RNA-seq, PMBCs were matched pools from 40 donors. For flow cytometry analysis, PBMCs were pooled from 5 donors. Cells were used after cryopreservation. T cells were activated in 24-well plates were pre-coated overnight at 4°C with 2 µg/mL rat a-mouse IgG2a (MW1483, Sanquin) in phosphate-buffered saline (PBS). Plates were washed with PBS and coated for >3 h with 1 µg/mL αCD3 (HIT3a, Biolegend) at 37°C. 1.3x106 PBMCs/well were seeded with 1 µg/mL soluble αCD28 (CD28.2, Biolegend) in 1 mL culture medium (Iscove's Modified Dubecco's Medium, IMDM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, 100 µg/mL streptomycin, and 2 mM L-glutamine. After 48h of activation at 37°C, 5% CO2, cells were harvested and cultured in standing T25/T75 tissue culture flasks (Thermo Scientific) at a density of 0.8x106/mL in culture medium supplemented with 100 IU/mL recombinant human (rh)IL2 (Protech) and 10 ng/mL rhIL-15 (Protech). Medium was refreshed every 2-3 days.
T cells were then restimulated using 1 μg/ml soluble α-CD3 (Pelicluster CD3, Sanquin) and 1 μg/mL soluble α-CD28 added to the culture media for the indicated durations. In the case of mTOR inhibition, 250nM Torin-1 (reconstituted in DMSO) was added during stimulation. In all cases, DMSO controls were included.
During RNA isolation and library preparation, ribosomal RNA (rRNA) was depleted. Approximately 30 x106 reads were obtained per sample. Sequencing adaptors trimming and low-quality read removal was performed using fastp [ref] (default settings. As rRNA depletion is not absolute, obtained reads were first mapped to a custom rRNA library (deposited to Zenodo) using STAR [ref]. Unmapped reads were subsequently aligned to the GRCh38 primary assembly using the gencode.v47 basic annotation (both files downloaded from https://www.gencodegenes.org/human). Aligned reads were subsequently quantified using htseq-count. Differential gene-expression analysis between indicated samples was performed using the R package limma.
