EXPERIMENT_TITLE: GSM5858317: CTCFKD_120h; Homo sapiens; Hi-C
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: Hi-C experiments were performed using MboI restriction enzyme as previously described (Rao, S. S. et al. A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping. Cell 159, 1665-1680, doi:10.1016/j.cell.2014.11.021 (2014)).  Briefly, RPE cells were crosslinked with 1% formaldehyde for 10 min at room temperature.  Cells were permeabilized and chromatin was digested with MboI restriction enzyme, and the ends of restriction fragments were labeled with biotinylated nucleotides and ligated.  After crosslink reversal, DNA was purified and sheared with Covaris M220 (Covaris).  Then point ligation junctions were pulled down with streptavidin beads. Sequencing libraries were constructed using Nextera Mate Pair Sample Preparation Kit (Illumina) according to the manufacturer's protocol with 15 PCR cycles.
STUDY_DESCRIPTOR_STUDY_ABSTRACT: Cohesin plays vital roles in chromatin folding and gene expression regulation, cooperating with such factors as cohesin loaders, unloaders, and the insulation factor CTCF. Although models of regulation have been proposed (e.g., loop extrusion), how cohesin and related factors collectively or individually regulate the hierarchical chromatin structure and gene expression remains unclear. We have depleted cohesin and related factors and then conducted a comprehensive evaluation of the resulting 3D genome, transcriptome and epigenome data. We observed substantial variation in depletion effects among factors at topologically associating domain (TAD) boundaries and on interTAD interactions, which were related to epigenomic status. Overall design: We used the in situ Hi-C protocol as described in Rao et al. (Rao et al., Cell, 2014, 10.1016/j.cell.2014.11.021). In brief, ~3 × 10^6 RPE cells were crosslinked with 1% formaldehyde for 10 min at room temperature, followed by an additional 5 min with 200 mM glycine in phosphate-buffered saline (PBS). Fixed cells were permeabilized in Hi-C lysis buffer (10 mM Tris-HCl, pH 8.0; 10 mM NaCl; 0.2% Igepal CA630; 1× protease inhibitor cocktail [Sigma]) on ice. The cells were treated with 100 U of MboI (New England Biolabs) for chromatin digestion, and the ends of digested fragments were labeled with biotinylated nucleotides followed by ligation. After DNA reverse crosslinking and purification, ligated DNA was sheared to a size of 300–500 bp using a Covaris S2 focused-ultrasonicator (settings: Duty Cycle, 10%; Intensity, 4; Cycles per Burst, 200; Duration, 55 sec). The ligated junctions were then pulled down with Dynabeads MyOne Streptavidin T1 beads (Thermo Fisher Scientific). The pulled-down DNA was end-repaired, ligated to sequencing adaptors, amplified on beads and purified using Nextera Mate Pair Sample Preparation Kit (Illumina) and Agencourt AMPure XP (Beckman Coulter). DNA was then sequenced to generate paired-end 150-bp reads using the Illumina HiSeq-2500 or X Ten system.
SAMPLE_TITLE: Hi-C siCTCF 120h
SAMPLE_SAMPLE_ATTRIBUTES_SAMPLE_ATTRIBUTE: [{"TAG": "source_name", "VALUE": "RPE cells"}, {"TAG": "enzyme", "VALUE": "MboI"}, {"TAG": "treatment", "VALUE": "CTCF siRNA#1"}, {"TAG": "time point", "VALUE": "120h"}]
Pool_Member_@attributes_sample_title: Hi-C siCTCF 120h
RUN_SET_RUN_Pool_Member_@attributes_sample_title: Hi-C siCTCF 120h
RUN_SET_RUN_SRAFiles_SRAFile: [{"Alternatives": null, "@attributes": {"cluster": "public", "filename": "CTCFKD_120h_R1.fastq.gz", "size": "39397973578", "date": "2022-02-04 07:32:39", "md5": "81d3e3f947a14257d59bb54f22b3594b", "version": "1", "semantic_name": "fastq", "supertype": "Original", "sratoolkit": "0"}}, {"Alternatives": null, "@attributes": {"cluster": "public", "filename": "CTCFKD_120h_R2.fastq.gz", "size": "41907339974", "date": "2022-02-04 07:46:00", "md5": "88da257bb91c81c5021bd389fa18f119", "version": "1", "semantic_name": "fastq", "supertype": "Original", "sratoolkit": "0"}}, {"Alternatives": null, "@attributes": {"cluster": "public", "filename": "SRR17870716", "url": "https://sra-pub-run-odp.s3.amazonaws.com/sra/SRR17870716/SRR17870716", "size": "55940444411", "date": "2022-02-04 08:23:09", "md5": "2b87de6f6d5ef2e7f8d88f5a0d830d89", "version": "1", "semantic_name": "SRA Normalized", "supertype": "Primary ETL", "sratoolkit": "1"}}, {"Alternatives": [null, null, null], "@attributes": {"cluster": "public", "filename": "SRR17870716.lite", "url": "https://sra-downloadb.be-md.ncbi.nlm.nih.gov/sos2/sra-pub-zq-18/SRR017/17870/SRR17870716/SRR17870716.lite.1", "size": "33414923167", "date": "2022-10-21 06:00:11", "md5": "bb1a8dc5a8fb0143973a3cead2806c6c", "version": "1", "semantic_name": "SRA Lite", "supertype": "Primary ETL", "sratoolkit": "1"}}]