GSM4972191: H3K4me3_ChRD_rep1; Oryza sativa; OTHER
About 5 g of samples were ground in liquid nitrogen into fine powder and then lysed in 7 ml 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) at 4 °C for 40 min with rotation at 12 rpm. The homogenate, containing the entire chromatin from the ground sample, was mixed with 30 ml of Buffer F (50 mM HEPES-KOH (pH7.5), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate), and Sonicate chromatin solution into 1–3 kb (High level, 36 cycles, 30 s ON, and 50 s OFF) using a Bioruptor (Diagenode). Centrifuge the chromatin fragments at 12,000 rpm for 10 min at 4 °C and transfer the supernatant to a new tube for chromatin immunoprecipitation (ChIP). To prepare antibody-loaded beads, 40 μl antibody (H3K4me3, ABclonal cat. no. A2357) was mixed with 750 μL suspended protein G magnetic beads (~1:100 dilution) followed by incubation at 4 °C for 8 h with rotation at 12 rpm. Then, mixed fragmented chromatin extract with antibody-loaded beads and incubated it at 4 °C for overnight with rotation at 12 rpm. The supernatant was discarded and the beads were washed using 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, then followed by washing with 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; the beads were washed with 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 finally followed washing with 5 mL 1× TE buffer (pH 8.0, Ambion, cat. no. AM9849) twice. DNA of 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 use specially designed single biotinylated bridge-linker: Forward strand: 5′-[5APP] CCTCGATAC/iBIOdT/TGAGCT GACT-3′, Reverse strand: 5′-[5Phos]GTCAGC/iBIOdT/CAAGTATCGAG -3′. Then the RNA ends were proximity-ligated by the single biotinylated bridge-linker with 5' adenylated on forward strand and followed by extending the Reverse strand in linker into ligated RNA with reverse transcription as first strand cDNA synthesis. Next, the DNA ends, with 5' A-tailing, were proximity-ligated by the single biotinylated bridge-linker with 3′ nucleotide T over-hanging on forward strand and followed by second strand cDNA synthesis. 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.