miCLIP profiling of m6A sites in human CD8? T cells during activation, with Input and noUV controls

Methylation individual-nucleotide-resolution crosslinking and immunoprecipitation (miCLIP) was performed in human primary CD8? T cells subjected to in vitro activation with aCD3/aCD28 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).