GSM6267966: young, shoots, Col-0, ChIA-PET, h3k27me3, rep1; Arabidopsis thaliana; ChIA-PET
About 5 g of tissues were used for each ChIA-PET assay. Tissues were ground to fine powder in liquid nitrogen and lysed in 10 ml of Buffer S (50mM HEPES-KOH (pH7.5), 150mM NaCl, 1mM 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 (50mM HEPES-KOH (pH7.5), 150mM NaCl, 1mM 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 was added to the supernatant for ChIP. ChIP was performed using antibodies against the following: H3K4me1 (ABclonal, A2355), H3K4me3 (ABclonal, A2357), H3K9me2 (Abcam, ab1220), H3K27me3 (ABclonal, A2363), and RNAPII (BioLegend, 920102). Antibody (50–80 mg) 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 PBST twice and incubated with sonicated chromatin overnight at 4°C with rotation. Subsequently, beads were washed two times for 5 min at 4°C in 5 ml of 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 of high-salt buffer (low salt ChIP buffer replaced 150 mM NaCl with 350 mM NaCl), one wash in 5 ml of 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 by using Tn5 transposase (VAHTS; cat. no. TD501). DNA fragments contained the bridge-linker at ligation junctions were captured by Dynabeads™ M-280 Streptavidin (Invitrogen, 11205D), and used as templates for PCR amplification.