GSM5133397: 120min_with_auxin_with_triptolide rep2; Mus musculus; Hi-C
In-situ Hi-C experiments were performed as previously described 1. Briefly, sorted cells (5 million for prometaphase and ana/telophase and 10million for early- and mid-G1 phase) were lysed in 1ml cold Cell Lysis Buffer (10mM Tris pH 8, 10mM NaCl, 0.2% NP-40/Igepal) for 10min on ice. Cells were pelleted at 4℃ and washed with 1.2 x DpnII buffer. Nuclei were permeabilized with 0.3% SDS for 1h at 37℃ and quenched with 1.8% Triton X-100 for 1h at 37℃. Chromatin was digested with 300U DpnII restriction enzyme (NEB, R0543M) in-situ at 37℃ over night with shaking. 300U DpnII restriction enzyme was added for an additional 4h at 37℃ with shaking. Nuclei were incubated at 65℃ for 20min to inactivate DpnII. After cool down, digested chromatin fragments were blunted with pCTP, pGTP, pTTP and Biotin-14-dATP (Thermal Fisher Scientific, 19524016) using 40U DNA Polymerase I, Large (Klenow) fragment (NEB, M0210). DNA was ligated in-situ with 4000U T4 DNA ligase (NEB, M0202M) for 4h at 16℃ followed by further incubation for 2h at RT. Nuclei were then incubated in 10% SDS containing proteinase K (3115879 BMB) at 65℃ overnight to reverse crosslinking. RNA was then digested with DNase-free RNase at 37℃ for 30min. DNA was then extracted by phenol-chloroform extraction, precipitated, and dissolved in nuclease free water. DNA was sonicated to 200-300bp fragments (Epishear, Active Motif, 100% amplitude, 30s ON and 30s OFF, 25-30min) and purified with AMPure XP beads (Beckman Coulter). For hic library construction, biotin-labeled DNA after extraction was purified by incubation with 100μl Dynabeads MyOne Streptavidin C1 beads (Thermal Fisher Scientific, 65002) at RT for 15min. DNA libraries were constructed using the NEBNext DNA Library Prep Master Mix Set for Illumina (NEB E6040, M0543L, E7335S). To elute DNA, streptavidin bead- bound DNA was incubated in 0.1% SDS at 98℃ for 10min. DNA was purified with AMPure XP beads and index labeled with NEBNext multiplex oligos for 6 cycles on a thermal cycler, using the NEBNext Q5 Hot Start HIFI PCR master mix. Index labeled PCR products were then purified with AMPure XP beads and sequenced on an Illumina NextSeq 500 sequencer.