Dpnii restriction enzyme

Nuclei were isolated from the fourth leaves of 4-week-old tomato plants using the same procedure as for the in situ Hi-C experiments. The Hi-ChIP protocol from Mumbach et al.^{68 (link)} was then applied using the DpnII restriction enzyme (NEB) and 3ug of anti-RNAPII antibody (Abcam, ab26721). The quality of the libraries was assessed using a Bioanalyzer (Agilent), and the libraries were subjected to 2 × 75 bp paired-end high-throughput sequencing by NextSeq 500 (Illumina).

In situ Hi-C was carried out as described^{5 (link)}. Cells were crosslinked with 1% formaldehyde then lysed to collect nuclei. Pelleted nuclei were digested with DpnII restriction enzyme (NEB, R0147). The restriction fragment overhangs were filled and marked with biotin-labelled dATP (Thermo Fisher, 19524016) and dCTP, dTTP, and dGTP before ligation. DNA was reverse crosslinked, purified, and fragmented by sonication on a Covaris sonicator. Biotin-labelled DNA was pulled-down on Streptavidin Dynabeads (NEB, S1420S). After DNA repair and 3′ A addition, SHORT Y-Adaptor (Supplementary Table 1) was added. Diluted DNA on Dynabeads was used for PCR amplification (4, 8, 12,16, and 20 cycles) to produce similar amounts of DNA for sequencing on the Illumina HiSeq X10 platform (paired end 2 × 150 bp reads).

Up to three million crosslinked cells were resuspended in 1000 µL of ice-cold cell lysis buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 0.2% NP-40, 1× Roche protease inhibitors) and rotated at 4 °C for 30 min. Nuclei were pelleted at 4 °C for 5 min at 1000 × g, and the supernatant was discarded. Pelleted nuclei were washed once with 500 µL of ice-cold cell lysis buffer. The supernatant was removed, and the nuclear pellet was resuspended in 50 µL of 0.5% SDS and incubated at 62 °C for 10 min. Then, 145 µL of water and 50 µL of 10% Triton X-100 were added, and the samples were rotated at 37 °C for 15 min to quench SDS. Then, 25 µL of 10x NEB Buffer 3.1 and 10 µL of 10 U/µL DpnII restriction enzyme (NEB, R0543T) were added, and the sample was rotated at 37 °C for 4 h. DpnII was then heat-inactivated at 62 °C for 20 min. Then, the reactions were rotated at 4 °C for 5 min. A total of 750 µL of ligation master mix was added: 100 µL of 10× NEB T4 DNA Ligase buffer with 10 mM ATP (NEB, B0202), 75 µL of 10% Triton X-100, 3 µL of 50 mg/mL BSA (Thermo Fisher, AM2616), 10 µL of 400 U/µL T4 DNA Ligase (NEB, M0202), and 562 µL of water. The reactions were rotated at 16 °C for 4 h and then allowed to proceed for an additional 1 h at RT.

About 1.5 g of 3-week-old seedlings were used for Hi-C experiment. The experiment procedures were similar to a previous study^{61 (link)} but some steps were improved for efficiency. To digest extra protein and make the nuclei more permeable, the nuclei were resuspended in 150 μl of 0.5% SDS buffer and incubated at 62 °C for 5 min. Chromatin was digested for 12 h with 20 units of DpnII restriction enzyme (NEB) at 37 °C and the resuspended mixture was incubated at 62 °C for 20 min to inactivate the restriction digestion. The DNA pieces between 300 and 500 bp were excised and purified using Ampure XP beads (Beckman Coulter). The library was constructed by an Illumina TruSeq DNA Sample Prep Kit and sequenced by Illumina Hiseq Xten with 2 × 150-bp reads.

For Hi-ChIP experiments, the nuclei were isolated from the shoots and roots using the same procedure as in the in situ Hi-C experiments, and the Hi-ChIP protocol from Mumbach et al. 2016 was then applied using the DpnII restriction enzyme (New England Biolabs) and the anti-polymerase II antibody (Active Motif, 39097). The quality of the libraries was assessed with Agilent 2100 Bioanalyzer (Agilent), and the libraries were subjected to 2 × 75 bp paired-end high-throughput sequencing by NextSeq 500 (Illumina).