Concanavalin a beads

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.