GSM7763685: DS_H3K9ac_rep1_IP; Oryza sativa; ChIP-Seq
Rice seedling strips were crosslinked with 1% formaldehyde (SIGMA, F8775) for 10–15 min at room temperature in a vacuum chamber, and 0.2 M glycine was applied to quench the reaction. For each reaction, ~0.1 g sample was ground in liquid nitrogen into fine powder and then lysed in 300 μL of Buffer S (50 mM HEPES-KOH (pH7.5), 150 mM NaCl, 1 mM Ethylene Diamine Tetraacetic Acid (EDTA), 1% Triton X-100, 0.1% sodium deoxycholate, 1% SDS) for 10 min at 4°C. The homogenate was mixed with 1.2 mL of Buffer F (50 mM HEPES-KOH (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate). After that, the chromatin in the homogenate was fragmented to 200–600 bp by sonication with a Bioruptor (Diagencode). Then, the mixture was centrifuged at 12000× g for 10 min at 4°C to recover the chromatin in the supernatant. 20 µL of the supernatant was reserved as input, and the remaining was used for ChIP. 50 μL Dynabeads® protein G beads (Life Technologies, 10003D) was balanced with PBST buffer (phosphate-buffered saline (PBS) with 0.1% Tween 20) twice before incubating with 1–2 μg anti-histone H3 acetyl K9 antibody (Abcam, ab10812) or previously approved anti-OsbZIP23 polyclonal antibody for 6–8 h on a rotator at 4°C. After removal of excessive antibody by washing with PBST twice, the antibody-conjugated beads were mixed with the sheared chromatin and incubated at 4°C overnight with gentle rotation. The immunoprecipitated chromatin was washed successively with low-salt ChIP buffer (50 mM HEPES-KOH, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, and 0.1% SDS) (three times), high-salt ChIP buffer (identical to low-salt ChIP buffer expect for the use of 350 mM NaCl) (twice), ChIP wash buffer (10 mM Tris-HCl pH 8.0, 250 mM LiCl, 0.5% NP-40, 1 mM EDTA, 0.1% sodium deoxycholate) (once), and TE buffer (10 mM Tris-HCl, pH 8.0, and 1 mM EDTA) (twice). The protein–DNA complex was eluted from the magnetic beads with 100 μL Elution Buffer (50 mM Tris-HCl pH 7.5, 10 mM EDTA, 1% SDS) at 65°C for 15 min and then reverse-cross-linked by overnight incubation with 20 mg/mL proteinase K and 5 M NaCl at 55°C. ChIP DNA libraries were prepared using the NEBNext® UltraTM II DNA library prep kit for Illumina® sequencing (New England BioLabs, E7645). Next, library DNA fragments with 250–650 bp in length were preserved using AMPure XP beads (Beckman, A63881).