Vavilovii_CENH3 (CRX1930302; CRA029290)
Nuclei were isolated from 3 to 5g of young rye leaves ground carefully in liquid nitrogen, using a lysis buffer containing 1 mM EDTA, protease inhibitor cocktail, 10 mM Tris-HCl (pH 7.5), 10 mM NaCl, 0.5% NP-40, 5 mM beta-ME, and 0.1 mM PMSF. Pelleted nuclei underwent two wash steps with lysis buffer and centrifugation at 1,200g for 5 min at 4C. The chromatin pellet resuspended in MNase buffer (10% sucrose, 50 mM Tris-HCl pH 7.5, 1 mM CaCl2, 1 mM MgCl2, 0.1 mM PMSF) and digested with 0.5 U MNase (New England BioLabs) at 37C for 20 min. EDTA quenched the reaction, followed by centrifugation at 12,000 rpm for 10 min at 4C. 50 uL protein A beads precleared the supernatant at 4C for 3-4 hours with rotation. An input sample separated, while the CENH3 or histone modification antibody incubated with the remainder overnight at 4C. Immunocomplexes recovered by 50 uL protein A beads underwent washes with low, medium, and high salt buffers. Elution occurred in buffer (50 mM NaCl, 20 mM Tris-HCl, 5 mM EDTA, 2% SDS) at 65C for 55 min.