GSM7763700: DS_WT_H3K9ac_ChIApet_rep2; Oryza sativa; ChIA-PET
Rice long-read ChIA-PET libraries were constructed with a modified ChIA-PET method. Tissues were dual cross-linked with 1% formaldehyde for 10 min and 1.5 mM ethylene glycol bis (succinimidylsuccinate) (Thermo Fisher Scientific, 21565) for 30 min at room temperature. After that, 0.2 M glycine was applied to halt the cross-linking reaction. Then, samples were ground in liquid nitrogen to a fine powder and approximately 5 g powder was lysed in 2× volume of buffer S. Next, each sample was applied with 8× volume of buffer F and sonicated in a Bioruptor® sonication system (Diagencode, UCD-200) to yield chromatin fragments with 1–3k bp in length. The chromatin fragments were collected by centrifuging at 12,000× g for 10 min at 4°C. 800 μL protein G with 25 μg anti-Histone H3 acetyl K9 antibody (Abcam, ab10812) was applied to the chromatin fragments and incubated at 4°C overnight with gentle rotation. The supernatant was discarded and the beads were sequentially washed with 5 mL low-salt ChIP buffer for three times, 5 mL high-salt ChIP buffer twice, 5 mL ChIP wash buffer once and 5 mL 1× TE buffer twice. ChIP DNA was used for end-repair and A-tailing using T4 DNA polymerase (Promega, cat. no. M421F) and Klenow enzyme (NEB, cat. no. M0212L). Proximity ligation of ChIP DNA was performed using biotinylated bridge-linker (forward strand: 5′-[5Phos] CGCGATATC/iBIOdT/TATCTGACT-3′, reverse strand: 5′-[5Phos] GTCAGA- TAAGATATCGCGT-3′). Protein-DNA complex was reverse cross-linked and the ChIA-PET libraries were prepared by using Tn5 transposase (VAHTS, TD501).