PLAC-seq procedure

PLAC-seq was performed as previously described⁵⁴. The frozen tissues were pulverized prior to formaldehyde crosslinking. About 30-50 mg of frozen tissue were crosslinked with 1% formaldehyde at room temperature for 20 min. Dissociation of crosslinked tissues were performed with gentleMACS dissociator. Single-nuclei suspensions prepared from crosslinked tissues were incubated in 50 μL 0.5% of SDS and incubated at 62 °C for 10 min. 25 μL 10% Triton X-100 and 145 μL water were then added, followed by incubation at 37 °C for 15 min. Digestion was performed by Mbol for 2 h 37 °C, followed by inactivation at 62 °C for 20 min. 15 nmol of dCTP, dGTP, dTTP, biotin-14-dATP (Thermo Fisher Scientific) each and 40 unit of Klenow were then added, and incubated at 37°C for 1.5h. Proximity ligation was performed at room temperature in 1X T4 DNA Ligase Buffer, 0.1 mg/ml BSA, 1% Triton X-100 and 4000 unit of T4 DNA Ligase (NEB). The nuclei were harvested at 2,500 g for 5 min and the supernatant was discarded. The nuclei were then resuspended in 130 μl RIPA buffer (10 mM Tris, pH 8.0, 140 mM NaCI, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.1% sodium deoxycholate, proteinase inhibitors) and lysed on ice for 10 min, followed by sonication using Covaris M220. The samples were centrifugation at 14,000 rpm for 20 min and supernatant was collected. The clear cell lysate was incubated with H3K4me3 antibody-coated (04-745, Millipore, 5 μg per sample) Dynabead M-280 Sheep Anti-Rabbit IgG at 4°C overnight. After incubation, the beads were washed with RIPA buffer three times, high-salt RIPA buffer (10 mM Tris, pH 8.0, 300 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.1% sodium deoxycholate) twice, LiCl buffer (10 mM Tris, pH 8.0, 250 mM LiCI, 1 mM EDTA, 0.5% IGEPAL CA-630, 0.1% sodium deoxycholate) once, TE buffer (10 mM Tris, pH 8.0, 0.1 mM EDTA) twice. To elute DNA, washed beads were first treated with 10 μg RNase A in extraction buffer (10 mM Tris, pH 8.0, 350 mM NaCl, 0.1 mM EDTA, 1% SDS) for 1 h at 37 °C, followed by adding 20 μg proteinase K and incubate at 65 °C 2 h. The fragmented DNA was purified by Zymo DNA Clean&Concentrator kit. Biotinylated DNA was pulled-down by Dynabeads MyOne Streptavidin T1 beads and PCR amplified for sequencing.