biostudies-arrayexpressJernej UleHomo sapienshttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-15649Glyoxal and nitrite-mediated deamination of unmethylated adenosines (GLORI) was applied to human primary CD8⁺ T cells subjected to in vitro activation with αCD3/αCD28 beads. To identify N⁶-methyladenosine (m⁶A) sites at single-nucleotide resolution and to test their dependence on METTL3, we generated GLORI libraries from non-activated (NoAct) CD8⁺ T cells, Day1-activated T cells treated for 24 hours with a competitive METTL3 inhibitor (4 μM), and Day5-activated T cells treated with the same inhibitor for 5 days. These datasets provide high-resolution m⁶A maps and allow comparison of m⁶A regulation in the presence or absence of METTL3 activity during distinct states of CD8⁺ T cell activation.biostudies-arrayexpressGrowth Protocol - 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.Nucleic Acid Extraction - 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).Sample Treatment - 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.Sample Collection - 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.Library Construction - 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.Sequencing - Libraries were sequenced on an Illumina NovaSeq 6000 by the Advanced Sequencing Facility at The Francis Crick Institute.OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesData Transformation - Sequencing reads were processed using GLORI-tools (https://github.com/liucongcas/GLORI-tools). After adapter trimming with Trim Galore (v0.6.7), reads were aligned to an A>G converted version of the human genome (GRCh38). Pileups were generated for all four nucleotides, and conversion ratios were calculated as A/(A+G) or T/(T+C). Sites were annotated using GenomicRanges and ChIPpeakAnno in R, with context assigned to promoters, exons, introns, and UTRs. Filtering thresholds included ≥15 reads per site and a conversion ratio ≥0.1. Processed datasets were saved as GRanges (.rds), and outputs included BigWig/BedGraph tracks, motif coordinates, and genomic distribution plots for downstream visualization and meta-analysis.UnknownTranscriptomicsGenomicsProteomicsIllumina NovaSeq 6000RNA-seq of coding RNAHomo sapiensJernej UlePaulo GameirofalseGLORI mapping of m⁶A sites in human primary CD8⁺ T cells under activation and METTL3 inhibitionGlyoxal and nitrite-mediated deamination of unmethylated adenosines (GLORI) was applied to human primary CD8⁺ T cells subjected to in vitro activation with αCD3/αCD28 beads. To identify N⁶-methyladenosine (m⁶A) sites at single-nucleotide resolution and to test their dependence on METTL3, we generated GLORI libraries from non-activated (NoAct) CD8⁺ T cells, Day1-activated T cells treated for 24 hours with a competitive METTL3 inhibitor (4 μM), and Day5-activated T cells treated with the same inhibitor for 5 days. These datasets provide high-resolution m⁶A maps and allow comparison of m⁶A regulation in the presence or absence of METTL3 activity during distinct states of CD8⁺ T cell activation.2025-10-15T00:00:00Z2026-04-13T18:01:44.201Z2025-11-11T17:07:08.5ZE-MTAB-15649ERP180862E-MTAB-15648E-MTAB-15643EFO_0002944EFO_0004170EFO_0003789EFO_0005518EFO_0003816EFO_0003738EFO_0004184EFO_0003969