biostudies-arrayexpressJernej UleHomo sapienshttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-15643Methylation individual-nucleotide-resolution crosslinking and immunoprecipitation (miCLIP) was performed in human primary CD8⁺ T cells subjected to in vitro activation with αCD3/αCD28 beads. To map m⁶A sites and assess their dynamic signatures during T cell activation, we applied an improved iCLIP (iiCLIP) protocol using an anti-m⁶A antibody (Abcam) on 750–100 ng of polyadenylated RNA. Samples were collected from non-activated (NoAct), Day 1-activated, and Day 5-activated CD8⁺ T cells isolated from healthy donors (NoAct: 4 donors; Day 1: 5 donors; Day 5: 4 donors). Two experimental controls were included: (i) “Input” libraries prepared without the m⁶A immunoprecipitation step (4 donors each for NoAct, Day 1, and Day 5), and (ii) “noUV” libraries generated without UV crosslinking (3 donors total).biostudies-arrayexpressGrowth Protocol - Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors (Cambridge Bioscience, NHSBT Cambridge, or Sanquin, NL) by Ficoll-Paque PLUS density gradient separation. Cells were maintained at 37 °C in 21% O₂ and 5% CO₂. CD8⁺ T cells were purified using MACS Miltenyi kits (cat. 130-096-495, 130-045-201, 130-059-901) following the manufacturer’s instructions.Sequencing - Libraries were sequenced on an Illumina HiSeq 2500 and HiSeq 4000 platform at the Advanced Sequencing Facility, The Francis Crick Institute.Library Construction - N6-methyladenosine (m⁶A) sites were profiled by miCLIP using the improved iiCLIP workflow. Fragmented RNA was immunoprecipitated with anti-m⁶A antibody (Abcam cat no. ab151230), UV crosslinked, ligated to infrared-labelled adapters, and separated by SDS–PAGE. After proteinase K digestion, RNA was reverse transcribed with Superscript IV using barcoded primers, cDNAs were bead-purified, circularised (CircLigase II), and amplified by PCR to generate sequencing-ready libraries. Input and noUV controls were processed in parallel without the IP or UV crosslinking steps.Sample Treatment - Purified CD8⁺ T cells were cultured in complete RPMI medium (10% FBS, 1% penicillin–streptomycin) supplemented with IL-2 (30 U/ml, Roche). For activation, cells were stimulated ex vivo with αCD3/αCD28 Dynabeads (Gibco, 11132D) at a 1:1 bead-to-cell ratio for either 1 or 5 days.Sample Collection - Primary human CD8⁺ T cells were isolated from peripheral blood mononuclear cells (PBMCs) obtained from healthy donors. Cells were either left non-activated (noAct) or activated with αCD3/αCD28 beads for 1 or 5 days, then spun down and stored at -80C prior to RNA extraction.Nucleic Acid Extraction - Total RNA was extracted using the mirVana kit (Thermo Fisher, #AM1560). Polyadenylated RNA (750–1000 ng) was purified by two rounds of oligo-dT bead capture (Thermo Fisher, #61002) and fragmented in buffered zinc acetate (pH 5.3) at 70 °C for 15 min.OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesData Transformation - The FlowBio iCLIP pipeline (https://docs.flow.bio/docs/clip-pipeline-1.0/) was used for preprocessing and mapping of miCLIP and Input libraries. Reads were demultiplexed, adapter-trimmed (TrimGalore), pre-mapped to rRNA/tRNA (Bowtie), and then aligned to the human GRCh38 genome with GENCODE annotation using STAR. Deduplication was performed with unique molecular identifiers (UMIs), and crosslink sites were defined as the read start minus one nucleotide. Peaks were called with Paraclu and Clippy. Quality control and crosslink counts summaries were generated in MultiQC reports. To identify enriched motifs, positionally enriched k-mer analysis (PEKA; https://github.com/ulelab/peka) was performed on high-confidence crosslinks within peaks, with parameters: -w 10, -dw 150, -k 5, -s 6, -p 0.7. PEKA scores were used to rank enriched k-mers (P < 0.01), and normalized crosslink distributions in the 3’UTR were used for downstream analyses. All processed files and reports are available via the FlowBio project portal (https://app.flow.bio/projects/663588932565340992/).MetabolomicsUnknownTranscriptomicsGenomicsProteomicsIllumina HiSeq 4000RNA-seq of coding RNAHomo sapiensJernej UlePaulo GameirofalsemiCLIP profiling of m6A sites in human CD8⁺ T cells during activation, with Input and noUV controlsMethylation individual-nucleotide-resolution crosslinking and immunoprecipitation (miCLIP) was performed in human primary CD8⁺ T cells subjected to in vitro activation with αCD3/αCD28 beads. To map m⁶A sites and assess their dynamic signatures during T cell activation, we applied an improved iCLIP (iiCLIP) protocol using an anti-m⁶A antibody (Abcam) on 750–100 ng of polyadenylated RNA. Samples were collected from non-activated (NoAct), Day 1-activated, and Day 5-activated CD8⁺ T cells isolated from healthy donors (NoAct: 4 donors; Day 1: 5 donors; Day 5: 4 donors). Two experimental controls were included: (i) “Input” libraries prepared without the m⁶A immunoprecipitation step (4 donors each for NoAct, Day 1, and Day 5), and (ii) “noUV” libraries generated without UV crosslinking (3 donors total).2025-11-01T00:00:00Z2026-05-27T12:30:41.755Z2025-09-30T22:51:07.46ZE-MTAB-15643ERP180841EFO_0002944EFO_0004170EFO_0003789EFO_0005518EFO_0003816EFO_0003738EFO_0004184EFO_0003969