GSM9758272: H3K27me3 coverage on SR2 -treated spheroids at 3 days-post treatment, replicate 1; Mus musculus; OTHER
CUT&Tag was performed using the Active Motif protocol (Active Motif #53160), following the manufacturer's specifications. Briefly, spheroids per sample were dissociated using Accutase, and 500,000 cells per sample were immobilized on concanavalin A-coated magnetic beads. Cells were sequentially incubated with 2 µg of primary antibodies against H3K4me3 (Sigma 17-614) or H3K27me3 (Abcam ab6002) overnight at 4°C with gentle rocking, followed by incubation with a-rabbit (H3K4me3) or a-mouse (H3K27me3) secondary antibodies  to enhance signal detection. Bead-bound cells were then incubated with a protein A–Tn5 transposase fusion protein preloaded with sequencing adapters. After targeted tagmentation (60 min at 37°C), DNA fragments were released by proteinase K digestion and purified using column-based cleanup for library preparation. Libraries were prepared using KAPA polymerase (Roche, KK2612) and single-index barcoded primers  as described in Buenrostro J et al 2025 . Library amplification cycles were determined by qPCR (CT + 2 for each sample). Libraries were purified using SPRI beads (CleanNA, # CNGS-0050), and quality was assessed by fluorometric quantification and fragment size analysis. Sequencing was performed on an Illumina NovaSeq X platform using 150 bp paired-end reads.