EXPERIMENT_TITLE: GSM4718289: MCM2-AID_Aux; Homo sapiens; Hi-C
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: Hi-C was performed largely as described in Rao et al, 2014 with minor modifications. Briefly, ~5x10 6 HCT116 cells were crosslinked in 1% formaldehyde for 10 min at RT, snap-frozen and stored at -80°C. After permeabilization in lysis buffer (0.2% Igepal, 10 mM Tris-HCl pH 8.0, 10 mM NaCl, 1x Halt Protease inhibitor cocktail) nuclei were isolated in 0.3% SDS in NEBuffer 3 at 62°C for 10 min. SDS was quenched with 1% Triton X-100 at 37°C for 1 h, then the nuclei were pelleted and resuspended in 250 μl DpnII buffer with 600 U DpnII (New England Biolabs) at 37˚C. After overnight digestion, 200 U DpnII were added for 2 h more incubation. Then, the ends were filled-in with d(G/C/T)TPs and biotin-14-dATP (Life Technologies) using Klenow (New England Biolabs) for 1.5 h at 37°C. After ligation at room temperature for 4 h with T4 ligase (New England Biolabs), the nuclei were spun down, resuspended in 200 μl mQ and digested with proteinase K for 30 min at 55°C in presence of 1% SDS. Cross-links were reversed at 65°C overnight after addition of NaCl to a final concentration of 1.85 M. After ethanol precipitation and a 70%–80% ethanol wash, DNA was resuspended in 50 µl of 10 mM Tris EDTA, incubated on ice for 15 min and then sheared to ~300-1300 bp fragments using the E220 Focused-ultrasonicator (Covaris). DNA was bound to MyOne C1 Streptavidin beads (ThermoFischer) and used for library preparation with the NEB Next Ultra Library Prep kit (Illumina). Libraries were sequenced on the NovaSeq 6000 system with 100 bp paired-end reads (at VBCF NGS Unit) The library preparation was done by the VBCF NGS unit (vbcf.ac.at) or MPIB NGS Core facility (https://www.biochem.mpg.de/7076201/NGS) following the standard DNA sample preparation protocol.
STUDY_DESCRIPTOR_STUDY_ABSTRACT: Eukaryotic genomes are compacted into loops and topologically associating domains (TADs), which contribute to transcription, recombination and genomic stability. Cohesin extrudes DNA into loops that are thought to lengthen until it encounters CTCF boundaries. Little is known whether loop extrusion is impeded by macromolecular machines. We demonstrate that the replicative helicase MCM is a barrier that restricts loops and TADs in G1 phase. Single-nucleus Hi-C of one-cell embryos revealed that MCM loading reduces CTCF-anchored loops and increases TAD boundary insulation, suggesting loop extrusion is impeded before reaching CTCF. Single-molecule imaging provides evidence that MCM are physical barriers that constrain cohesin translocation in vitro. Simulations are consistent with MCM as abundant, random barriers with low permeability. We conclude that distinct loop extrusion barriers contribute to shaping 3D genomes. Overall design: in situ Hi-C and micro-C from HCT116 MCM2-AID cells, treated with DMSO or Auxin, and with or without triptolide treatment
SAMPLE_TITLE: MCM2-AID_Aux
SAMPLE_SAMPLE_ATTRIBUTES_SAMPLE_ATTRIBUTE: [{"TAG": "source_name", "VALUE": "HCT116 MCM2-AID"}, {"TAG": "genotype", "VALUE": "MCM2-AID"}, {"TAG": "treatment", "VALUE": "Auxin"}]
Pool_Member_@attributes_sample_title: MCM2-AID_Aux
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