GSM5627231: H3_ChRD_rep2; Oryza sativa; OTHER
About 3 g Rice nuclei isolated from dual cross-linked rice MH63 seedlings were  lysed in Buffer S  (50 mM HEPES-KOH (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, and 1% SDS) at 4 °C for 40 min at 12 rpm. Then add 9 volume buffer F (50 mM HEPES-KOH (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, and 0.1% sodium deoxycholate) to sample and rotate at 4 °C for 40 min at 12 rpm. The chromatin were then sonicated into 1–3 kb (High level, 28 cycles, 30 s ON, and 50 s OFF) using a Bioruptor (Diagenode). The chromatin fragments were centrifuged at 2800 g for 10 min at 4 °C and the supernatant was transferred to a new tube for chromatin immunoprecipitation (ChIP). To prepare antibody-loaded beads, 40 μL antibody (H3K4me3 antibody, ABclonal cat. no. A2357；H3 antibody, ABclonal cat. no. A2348) was mixed with 750 μL of suspended protein G magnetic beads, followed by incubation at 4 °C for 8 h with rotation at 12 rpm. Then, the mixed fragmented chromatin extract was incubated with antibody-loaded beads and incubated at 4 °C overnight with rotation at 12 rpm. The supernatant was discarded and the beads were washed three times with 1 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), and then washed twice with 1 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); and once with 1 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), and finally washed with 1 mL 1× TE buffer (pH 8.0, Ambion, cat. no. AM9849) twice. DNA of the ChIP complex on beads was used for end-repair and A-tailing using T4 DNA polymerase (Promega, cat. no. M421F), and Klenow enzyme (NEB, cat. no. M0212L). To ligate DNA and RNA, we used a specially designed single biotinylated bridge linker: forward strand: 5′-[5APP]CCTCGATAC/iBIOdT/TGAGCTGACT-3′; reverse strand: 5′-[5Phos]GTCAGC/iBIOdT/CAAGTATCGAG-3′. Then, RNA ends were proximity-ligated by the single biotinylated bridge-linker with the 5′ end adenylated on the forward strand, followed by extension of the reverse strand in the linker into ligated RNA with reverse transcription as first strand cDNA synthesis. Next, the DNA ends with 3' A-tailing were proximity-ligated by the single biotinylated bridge linker with a 3' nucleotide T over-hang on the forward strand, followed by second strand cDNA synthesis. (For RNase H treated H3K4me3 ChRD-PET data, RNase H treatment was conducted before DNA end repair and A-tailing.) The proximity ligation RNA-DNA fragments were reverse cross-linked and fragmented before adding sequencing adaptors simultaneously by using Tn5 transposase (VAHTS, cat. no. TD501). The fragments contained the bridge-linker at ligation junctions were captured by Streptavidin beads, and used as templates for PCR amplification. These DNA products were then subjected to size-selection and paired-end sequencing (2 × 150 bp) using Illumina Hiseq X-Ten.