DMSO_Day1-activated_A
Homo sapiens
Protocols: Peripheral blood mononuclear cells (PBMCs) were obtained from healthy donors through Cambridge Bioscience, NHS Blood and Transplant (UK), or Sanquin (Netherlands). Ethical approval and written informed consent were obtained in accordance with the Declaration of Helsinki (2013). PBMCs were isolated using Ficoll-Paque PLUS density gradient centrifugation, and CD8⁺ T cells were purified using the MACS Miltenyi CD8⁺ MicroBeads kit (cat. no. 130-045-201). Cells were either used fresh (8–12 h post-blood collection) or thawed from cryopreservation. CD8⁺ T cells were cultured in complete RPMI-1640 medium (Gibco, 52400-025) supplemented with 10% foetal bovine serum (FBS), 1% penicillin–streptomycin, and 30 U/ml recombinant human IL-2 (Clinigen). Cultures were maintained at 37 °C with 21% O₂ and 5% CO₂. Cells were either left non-activated (Day 0) or activated with anti-CD3/CD28 Dynabeads (Gibco, 11132D) at a 1:1 bead-to-cell ratio for 1 or 5 days. Non-activated and Day1 activated cells were treated for 24 hours with DMSO or 4 µM METTL3 inhibitor (MedChemExpress, cat. no. HY-134836), while Day5 activated T cells were treated with the same inhibitor for 5 days. For Day 5 activated samples, beads were removed at day 4 and fresh medium was replenished twice during culture. CD8⁺ T cell pellets were lysed in TRIzol, and total RNA was isolated using Direct-zol RNA Miniprep Kits (Zymo Research, cat. no. R2052) according to the manufacturer's instructions. RNA quality was assessed with a Bioanalyzer (Agilent). GLORI libraries were prepared using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs, cat. no. E7760S). The protocol uses a dUTP-based method for second-strand synthesis, resulting in strand-specific libraries in which Read 2 corresponds to the sense strand of the original RNA. Library preparation was carried out according to the manufacturer's instructions.
ENA first public
2025-10-30
INSDC center name
Dementia Research Institute at KCL, London, UK The Francis Crick Institute, London, UK National Institute of Chemistry, Ljubljana, Slovenia
INSDC status
public
Submitter Id
E-MTAB-15649:DMSO_Day1-activated_A
broker name
ArrayExpress
cell type
CD8-Positive T-Lymphocytes
collection date
not collected
common name
human
genotype
wild type genotype
geographic location (country and/or sea)
not collected
isolate
not applicable
replicate
1
sample name
E-MTAB-15649:DMSO_Day1-activated_A
scientific_name
Homo sapiens