2025-10-30
ENA first public
ERC000011
ENA-CHECKLIST
Dementia Research Institute at KCL, London, UK The Francis Crick Institute, London, UK National Institute of Chemistry, Ljubljana, Slovenia
INSDC center name
public
INSDC status
ERS26957639
SRA accession
E-MTAB-15649:METTL3-in_Day1-activated_A
Submitter Id
ArrayExpress
broker name
CD8-Positive T-Lymphocytes
cell type
not collected
collection date
human
common name
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.
description
wild type genotype
genotype
not collected
geographic location (country and/or sea)
not applicable
isolate
Homo sapiens
organism
Homo sapiens
organism
1
replicate
Homo sapiens
scientific_name
METTL3-in_Day1-activated_A
title