title: miCLIP_non-Activated_R3
organism: Homo sapiens
description: Protocols: 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. 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. 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. 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. 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.