KY21 CENH3 ChIP-seq
Plant material was ground to a fine powder in liquid nitrogen using mortar and pestle. The powder was cross-linked with 1% formaldehyde in cross-linking buffer (0.4 M sucrose; 10 mM Tris-HCl, pH 8.0; 1 mM EDTA; 1 mM PMSF) for 20 minutes on ice, and cross-linking was stopped by adding 0.1 M glycine (final concentration) for another 5 minutes on ice. After filtering through two layers of miracloth, the crude nuclei were isolated using M1 (11.9% hexylene glycol; 10 mM KPO4, pH 7.0; 100 mM NaCl; 5 mM beta-mercaptoethanol; 0.1 mM PMSF, plant protease inhibitor cocktail) and M2 buffer (8.85% hexylene glycol; 10 mM KPO4, pH 7.0; 10 mM MgCl2; 0.5% Triton X-100; 5 mM beta-mercaptoethanol; 100 mM NaCl). Chromatin in the crude nuclei preparation was digested with Micrococcal Nuclease in MNB buffer (50 mM Tris-Cl, pH 8.0; 1 mM CaCl2; 4 mM MgCl2; 0.3 M sucrose) at 37°C to produce mono- and oligonucleosomes. After clearing with protein A dynabeads (Invitrogen, 100-02D), the chromatin was incubated with purified anti-ZmCenH3 antibody. Rabbit IgG antibody was included as a negative control. After overnight incubation by rotating in the cold room, the antibody-chromatin complex was immuno-precipitated with protein A dynabeads, followed by washing, elution, reverse cross-link and DNA purification. After ChIP quality was confirmed, 10 ng of ChIPed DNA were used for Illumina sequencing (University of Utah).
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