GSM7429886: young_shoots_Col-0_ChIA-PET_2_H3K4me3_rep2; Arabidopsis thaliana; OTHER
About 2 g of sample was used for each ChIA-PET assay. Tissues were ground to fine powder in liquid nitrogen and lysed in 10 ml of Buffer S (50 mM HEPES-KOH (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 1% SDS) for 20 min at 4 °C. The homogenate was mixed with 20 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), and the chromatin was fragmented into 1–3 kb by sonication using a Bioruptor (Diagenode). The lysates were centrifuged at 20,000 × g for 5 min at 4 °C and 20 ml of Buffer F were added to the supernatant for ChIP. ChIP was performed using antibodies against the following: H3K4me3 (ABclonal, A2357). Antibody (50–80 g) was added to Dynabeads® protein G beads (Life Technologies, 10003D) and incubated for 6 h on a rotator at 4 °C. Beads were washed with phosphate-buffered saline with Tween® (PBST) twice and incubated with sonicated chromatin overnight at 4 °C with rotation. Subsequently, beads were washed twice for 5 min each at 4 °C in 5 ml low-salt buffer (50 mM HEPES-KOH, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS), followed by two washes in 5 ml high-salt buffer (low salt ChIP buffer in which 150 mM NaCl was replaced with 350 mM NaCl), one wash in 5 ml LiCl wash buffer (10 mM Tris-HCl pH 8.0, 250 mM LiCl, 0.5% NP-40, 1 mM EDTA, 0.1% sodium deoxycholate), and one wash in TE buffer (10 mM Tris-HCl, pH 8.0, and 1 mM EDTA). ChIP DNA 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). 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′). Proximity ligation DNA was reverse cross-linked, and the ChIA-PET libraries were prepared using Tn5 transposase (VAHTS; cat. no. TD501). DNA fragments containing the bridge-linker at ligation junctions were captured by Dynabeads™ M-280 Streptavidin (Invitrogen, 11205D), and used as templates for PCR amplification.