title: GSM9329862: Ctrl; Homo sapiens; OTHER
library_construction_protocol: CUT&Tag libraries for Pol II were generated from human intestinal organoids exposed to vehicle or Listeria monocytogenes (3 biological replicates). Nuclei were isolated from Listeria-infected and control human intestinal organoids by gentle douncing in NE1 buffer. CUT&Tag was performed on 100,000 nuclei per sample following the established protocol. Nuclei were immobilised on concanavalin A-coated magnetic beads and incubated overnight at 4°C with anti-RPB1 (Pol II) antibody (CST 2629) in digitonin-containing antibody buffer. After washing, a mouse secondary antibody and pre-loaded pAG-Tn5 adapter complex (EpiCypher) were sequentially applied, followed by tagmentation at 37°C for 1 h. DNA was released by heat in SDS buffer, neutralized, and purified by spin columns. Libraries were generated using NEBNext High-Fidelity PCR Master Mix and Nextera i5/i7 indexed primers, amplified for 11 cycles, size-selected with AMPure beads, quantified, assessed on a TapeStation, pooled equimolarly, and sequenced on the NovaSeq X Plus (PE150; ~20 million reads/sample)