EXPERIMENT_TITLE: GSM4613670: Hi-C: G1 control + 4sT; Homo sapiens; Hi-C
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: For Hi-C, fixed cells were permeabilized using ice-cold Hi-C lysis buffer (10 mM TRIS-HCl pH 8 [Sigma], 10 mM NaCl [Sigma], 0.2 % Nonidet P-40 substitute [Sigma], 1x Complete EDTA-free Protease inhibitor [Roche]) for 30 min at 4 °C. Then, cells were spun down (2500 x g for 5 min), supernatant was discarded, and digestion mix was added (375 U DpnII [NEB] in 1x DpnII buffer [NEB]) and cells incubated for 16 h at 37 °C under rotation. Then, cells were spun down, supernatant was discarded and fill-in mix was added (38 µM Biotin-14-dATP [Thermo Fisher Scientific], 38 µM dCTP, dGTP and dCTP [Thermo Fisher Scientific], 50 U Klenow Polymerase [NEB], 1x NEB 2 buffer) and cells incubated for 1 h at 37 °C under rotation. Then, cells were spun down again, and ligation mix was added (1x T4 DNA ligase buffer [Thermo Fisher Scientific], 0.1 % Triton X-100 [Sigma], 100 µg/ml BSA [Sigma], 50 U T4 DNA ligase [Thermo Fisher Scientific]) and incubated at RT for 4 h. Then, cells were spun down, resuspended in 200 µl PBS and gDNA was purified using the DNeasy Blood and Tissue kit (Quiagen). DNA was transferred to a Covaris microTUBE (Covaris) and sheared on a Covaris S2 instrument (Duty cycle 10 %, Intensity 5.0, Cycles/burst 200) for 25 s. Double size selection was performed by employing AMPure XP beads (Beckman Coulter) first at 0.8-fold sample volume according to the standard protocol, followed by transfer of the supernatant and bead application at 0.12-fold sample volume. The resulting DNA was bound to Dynabeads MyOne Streptavidin C1 beads (Thermo Fisher Scientific) in Biotin binding buffer (5 mM Tris-HCl pH 7.5 [Sigma], 0.5 mM EDTA [AppliChem], 1 M NaCl [Merck]) for 1 h at RT. Beads were then washed 2x in Tween wash buffer (5 mM Tris-HCl [Sigma], 0.5 mM EDTA [AppliChem], 1 M NaCl [Merck], 0.05 % Tween-20 [Sigma]) and 1x in H2O. For DNAseq, Cells were harvested by trypsinization, spun down at 1100 x g for 1 min and the supernatant was discarded. Then, cells were spun down, resuspended in 200 µl PBS and gDNA was purified using the DNeasy Blood and Tissue kit (Quiagen). DNA was transferred to Covaris microTUBE (Covaris) and sheared on a Covaris S2 instrument (Duty cycle 10 %, Intensity 5.0, Cycles/burst 200) for 25 s. Double size selection was performed by employing AMPure XP beads (Beckman Coulter) first at 0.8-fold sample volume according to the standard protocol, followed by transfer of the supernatant and bead application at 0.12-fold sample volume. For Hi-C samples, beads with bound biotinylated DNA were resuspended in H2O and library preparation was performed using the NEBNext Ultra II DNA library prep kit for Illumina [NEB] according to the standard protocol. After this, beads were washed 4x using Tween wash buffer and DNA was eluted using 95 % formamide (Sigma), 10 mM EDTA (AppliChem) at 65 °C for 2 min. DNA was then precipitated using 80 % EtOH (Sigma), washed with 75 % EtOH and resuspended in H2O. Then, 4sT was converted to methyl-cytosine using OsO4 / NH4Cl, followed by qPCR according the NEBUltra Ultra II DNA library prep kit for Illumina [NEB]. The finished libraries were purified using AMPure XP beads (Beckman Coulter) at 0.9x sample volume following the standard protocol. For DNAseq samples, DNA library preparation was performed using the NEBNext Ultra II DNA library prep kit for Illumina [NEB] according to the standard protocol. The unamplified libraries were then treated using OsO4  and amplified according the NEBNext Ultra II DNA library prep kit for Illumina [NEB]. The finished libraries were purified using AMPure XP beads (Beckman Coulter) at 0.9x sample volume following the standard protocol.
STUDY_DESCRIPTOR_STUDY_ABSTRACT: The three-dimensional organization of the genome supports regulated gene expression, recombination, DNA repair, and chromosome segregation during mitosis. Chromosome conformation capture (Hi-C) has revealed a complex genomic landscape of internal chromosome structures in vertebrate cells yet how sister chromatids topologically interact in replicated chromosomes has remained elusive due to their identical sequences. Here, we present sister-chromatid-sensitive Hi-C (scsHi-C) based on nascent DNA labeling with 4-thio-thymidine. Genome-wide conformation maps of human chromosomes revealed that sister chromatid pairs interact most frequently at the boundaries of topologically associating domains (TADs). Continuous loading of a dynamic cohesin pool separates sister-chromatid pairs inside TADs and is required to focus sister chromatid contacts at TAD boundaries. We identified a subset of TADs that are overall highly paired, characterized by facultative heterochromatin, as well as insulated topological domains that form separately within individual sister chromatids. The rich pattern of sister chromatid topologies and our scsHi-C technology will make it possible to dissect how physical interactions between identical DNA molecules contribute to DNA repair, gene expression, chromosome segregation, and potentially other biological processes. Overall design: Sister-chromatid sensitive Hi-C (scsHi-C) Method validation using DNAseq and analysis of chromatin structure by scsHi-C with and without protein depletion using the auxin-inducible degron (AID) systems.
SAMPLE_TITLE: Hi-C: G1 control + 4sT
SAMPLE_SAMPLE_ATTRIBUTES_SAMPLE_ATTRIBUTE: [{"TAG": "source_name", "VALUE": "cervical carcinoma"}, {"TAG": "cell type", "VALUE": "HeLa Kyoto"}, {"TAG": "diesease state", "VALUE": "cervical carcinoma"}, {"TAG": "genotype", "VALUE": "w.t. G1"}]
Pool_Member_@attributes_sample_title: Hi-C: G1 control + 4sT
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