GSM4273165: RNAPII_ChIA_20h_rep2; Oryza sativa; OTHER
Sample was ground in liquid nitrogen into a fine powder and resuspended in 100 ml of EB1 buffer. The mixture was then filtered through Miracloth, and the filtrate was centrifuged at 1,800 × g for 10 min at 4 °C. The pellet was washed three times in 5 ml of EB2 buffer and centrifuged at 2,000 × g for 10 min at 4 °C. Next, the pellet was washed in 2 ml of EB3 buffer and centrifuged at 2,000 × g for 1 h at 4 °C. The final pellet was resuspended in 1 ml of NLB buffer, and chromatin was fragmented into 1-3 kb by sonication. After centrifugation at 2,000 × g for 10 min at 4 °C, the supernatant was transferred to a new tube for chromatin immunoprecipitation (ChIP). The antibody (80 µg; RNAPII, BioLegend, 920102) was mixed with 800 µl suspended protein G magnetic beads, and incubated at 4°C for 8 h with rotation to coat the magnetic beads. All of the chromatin was extracted to the antibody-loaded beads in a new tube and incubated at 4°C overnight with rotation. The supernatant was discarded and the beads were sequentially washed with 5 mL 0.1% SDS FA cell lysis buffer (0.05 M HEPES-KOH, 0.15 M NaCl, 0.001 M EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, and 0.1% SDS) for three times, 5 mL High salt ChIP buffer (0.05 M HEPES-KOH, 0.35 M NaCl, 0.001 M EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, and 0.1% SDS) twice, 5 mL ChIP wash buffer (0.01 M Tris–HCl, 0.25 M LiCl, 0.001 M EDTA, 2.5% NP-40, and 0.5% sodium deoxycholate) once and 5 mL 1 × TE buffer (pH 8.0, Ambion, cat. no. AM9849) 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]GTCAGATAAGATATCGCGT-3′). Protein-DNA complex was reverse cross-linked and the ChIA-PET libraries were prepared by using Tn5 transposase (VAHTS; cat. no. TD501). The libraries were sequenced (2 × 150 bp) using Illumina Hiseq X-Ten (Beijing AnnoRoad Company).
GEO Accession
GSM4273165