Concanavalin a coated magnetic beads

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 (link)]. 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 GSE190248.

CUT&RUN experiments were carried out as previously described (Skene et al., 2018 (link)) with the following modifications: 1–2.5×10⁵ cells were isolated by FACS as described in sections above, bound to Concanavalin A coated magnetic beads (Bangs Laboratories), and permeabilized with 0.025% (wt/vol) digitonin. Permeabilized cells were incubated overnight at 4°C with 5ug of anti-H3K27me3 (Active Motif) and then washed before incubating with protein A-MNase fusion protein (a gift from S. Henikoff) for 15 minutes at room temperature. After washing, cells were incubated in CaCl₂ to induce MNase cleavage activity for 30 minutes at 0°C. The reaction was stopped with 2xSTOP buffer (200 mM NaCl, 20 mM EDTA, 4 mM EGTA, 50 μg/mL RNase A, 50 μg/mL glycogen, and 2pg/mL of yeast spike-in DNA). Histone-DNA complexes were isolated from insoluble nuclear chromatin by centrifugation and DNA was extracted with a NucleoSpin PCR Clean-up kit (Macherey-Nagel). For CUT&RUN quantitative PCR, human Kasumi-1 cell line (ATCC CRL-2724) were added before binding the cells to Concanavalin A beads for internal standard instead of yeast spike-in DNA.

Adult CMs were isolated from 10- to 12-week-old mice by Langendorff perfusion. The CM fraction was enriched by low-speed centrifugation at 50g for 5 minutes. H3K27ac CUT&RUN was performed in CMs as previously described (57 (link)). A total of 200,000 CMs were used for each CUT&RUN experiment. H3K27ac (Cell Signaling Technology, catalog 8173) or rabbit IgG (Cell Signaling Technology, catalog 3900) at 1:100 was incubated with CMs, which were attached to concanavalin A–coated magnetic beads (Bangs Laboratories, catalog BP531) overnight at 4°C. pA-MNase (2.5 μL) from a 1:10 dilution of the original stock provided by the Steven Henikoff laboratory at the Fred Hutch Cancer Center was applied to the cell-bead mixture and incubated at room temperature for 10 minutes. Pull-down DNA was extracted in phase-lock tubes using the phenol-chloroform method. The DNA library was prepared using the Hyper-Prep kit (KAPA Biosystems KK8502) and submitted to the Next-Generation Sequencing Core at the University of Pennsylvania for sequencing. PE sequencing at 40 bp × 40 bp was adopted.

CUT&RUN was performed as described (27 (link)). Briefly, 500 000 cells were washed with Wash Buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine and protease inhibitors), bound to Concanavalin A-coated magnetic beads (Bangs Laboratories) and incubated with SOX9 antibody (1:100, AB5535, Millipore) diluted in wash buffer containing 0.05% digitonin (Dig-Wash) overnight at 4°C. Cells were washed and incubated with Guinea Pig anti-Rabbit secondary antibody (1:100, Novus Biologicals) diluted in Dig-Wash for 1 h at RT, washed again and incubated with CUTANA™ pAG-MNase (Epicypher) for 10 min at RT. Slurry was washed, placed on ice and incubated with Dig-Wash containing 2 mM CaCl₂ for 30 min to activate digestion. Stop buffer (340 mM NaCl, 20 mM EDTA, 4 mM EGTA, 0.05% Digitonin, 0.05 mg/ml glycogen, 5 μg/ml RNase A) was added, and fragments were released by 30 min incubation at 37°C. DNA was extracted with phenol-chloroform and ethanol precipitation. Libraries were constructed and sequenced. Two biological replicates of SOX9 at 96 h and one replicate of the SOX9 time-course were sequenced.