ChIP-sequencing (ChIP-seq) analysis and quantitative ChIP (qChIP)

ChIP-seq analysis was performed using cleavage under targets and tagmentation (CUT&Tag, NOVOPROTEIN, Shanghai, China). All procedures strictly followed the kit instructions, as described previously [27]. Briefly, 1 × 10⁶ MDA-MB-231 cells were washed and bound with concanavalin A-coated magnetic beads (Bangs Laboratories, IN, USA). The bead-bound cells were resuspended in 50–100 µL dig-wash buffer and incubated with 4 μg anti-MTA1 and anti-RUNX2 primary antibodies. The primary antibody was removed, followed by incubation with the secondary antibody. Next, cells were resuspended in Tagmentation buffer and incubated at 37 °C for 1 h. Ampure XP beads were added to each tube by vortexing, and quickly spun to extract the DNA. Beads were washed and eluted using 30–40 µL of 10 mM Tris at pH 8. The elution liquid was used for library construction and high-throughput sequencing. For the qChIP assay, 1 × 10⁷ MDA-MB-231 cells were cross-linked with 1% formaldehyde, sonicated, pre-cleared, and incubated with 4 μg of antibody per reaction. Complexes were washed with low-and high-salt concentration buffers, and the DNA was extracted for qChIP assay using the QIAquick PCR Purification Kit. The specific primers used for the qPCR assay were supplied in Supplementary Table S4. ChIP-seq results are available on {"type":"entrez-geo","attrs":{"text":"GSE190248","term_id":"190248"}}GSE190248.