EXPERIMENT_TITLE: GSM2978429: HiC_UNC4976_4h; Homo sapiens; Hi-C
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: For HiC experiments, 5-10 million purified CD4+ T lymphocytes were fixed for 10 min with 1 % formaldehyde after 4h treatment with 20 mM UNC4976 (0.2% DMSO) or 0.2% DMSO. The fixing was quenched by adding glycine (100 mM final concentration). After pelleting at 1200g and decanting, cells were resuspended with 10 mL 3e Hi-C lysis buffer (10 mM Tris-HCl pH 8.0, 10 mM NaCl, 0.2% NP40, and Sigma protease inhibitor cocktail) and incubated at 4 °C for 1 h. The cells were pelleted and resuspended with 400 mL CutSmart Buffer (1X, NEB), adjusted to 0.1% SDS, and heated at 65 °C for 10 min. The mixture was treated with 10 % Triton X-100 (44 mL), and digested with CviQI (20U, NEB) and CviAII (20U, NEB) at room temperature for 20 min. Bfa I was added (20U, NEB) and the mixture was incubated at 37 °C for 20 min. The reaction mixture was quenched with the addition of 600 mL wash buffer (10mM NaCl, 1mM EDTA, 0.1% Triton X-100). The cells were pelleted, and washed again with 600 mL wash buffer. Blunt-ended DNA fragments were generated by incubating with Klenow Fragment (NEB) for ligation of dCTP, dGTP, dTTP, biotin-dATP, followed by blunt-end ligation with T4 DNA ligase (NEB) at 16 °C overnight. The samples were de-crosslinked, treated with T4 DNA Polymerase, and sheared to a median fragment length of 400 bp with Diagenode sonicator. After DNA End Repair and A-tailing according to standard procedure31 and biotin capture was performed with Streptavidin C1 beads (ThermoFisher) according to manufacturer's specifications. On-bead ligation was carried out with Paired-End Adaptors (Illumina) and PCR amplification and size selection afforded indexed sequencing libraries with a median fragment length of 500 bp. After 2% E-gel purification (ThermoFisher), libraries were paired-end sequenced on the HiSeq 2500.
STUDY_DESCRIPTOR_STUDY_ABSTRACT: While the repressive roles of Polycomb complexes have been well-established with in vitro systems, it remains unclear how subtypes of these complexes function in living cells. Conditional alleles have enabled studies of Polycomb Repressive Complex 1 (PRC1), but the available timescales on which inducible deletion occurs is not sufficient to define its immediate regulatory targets. We have used rapid treatments with a new PRC1 inhibitor UNC4976, that functions as a chemical surrogate for H3K27me3 and engages chromodomain-containing subunit CBX7. This “histone prosthetic” inhibition strategy has enabled the discovery that hundreds of chromatin loop and contact domains are repressed by the chromodomain function of PRC1. Repressive chromatin architecture that is dependent specifically upon regulatory function of the PRC1 chromodomain is rapidly de-repressed with chemical inhibition within 4 hours. With Hi-C experiments after rapid drug treatment, and defining drug-sensitive chromatin by ChIP-seq before major transcriptional changes occur, we report canonical PRC1-dependent chromatin architecture in human T lymphocytes. Overall design: Measuring effects of UNC4976 on CBX7 binding, gene expression, chromatin accessibility and chromatin interactions on human CD4 T cells by applying CBX7 ChIP-Seq, RNA-Seq, ATAC-Seq, HiC experiments in human CD4 T cells with treatment of UNC4976 for 4 hours.
SAMPLE_TITLE: HiC_UNC4976_4h
SAMPLE_SAMPLE_ATTRIBUTES_SAMPLE_ATTRIBUTE: [{"TAG": "source_name", "VALUE": "Lymphocytes"}, {"TAG": "cell type", "VALUE": "CD4+ T cells"}, {"TAG": "treatment condition", "VALUE": "UNC4976 (4h)"}]
Pool_Member_@attributes_sample_title: HiC_UNC4976_4h
RUN_SET_RUN_Pool_Member_@attributes_sample_title: HiC_UNC4976_4h
RUN_SET_RUN_SRAFiles_SRAFile: [{"Alternatives": [null, null, null], "@attributes": {"cluster": "public", "filename": "SRR6674654.sralite", "url": "https://sra-downloadb.be-md.ncbi.nlm.nih.gov/sos5/sra-pub-zq-14/SRR006/674/SRR6674654.sralite.1", "size": "8671129774", "date": "2020-06-20 11:19:32", "md5": "8db5fbfa1ae08927af1149a23050ffea", "version": "1", "semantic_name": "SRA Lite", "supertype": "Primary ETL", "sratoolkit": "1"}}, {"Alternatives": null, "@attributes": {"cluster": "public", "filename": "SRR6674654", "url": "https://sra-pub-run-odp.s3.amazonaws.com/sra/SRR6674654/SRR6674654", "size": "13285773058", "date": "2018-02-05 17:59:55", "md5": "5b5ce5410f78706a5a6b2363dec4ea0e", "version": "1", "semantic_name": "SRA Normalized", "supertype": "Primary ETL", "sratoolkit": "1"}}, {"Alternatives": null, "@attributes": {"cluster": "public", "filename": "KZ1354_GA9939_R2.fastq.gz", "size": "11987930613", "date": "2020-03-05 18:26:59", "md5": "e5fe9e44560c68a5e8498de91d19b4d0", "version": "1", "semantic_name": "fastq", "supertype": "Original", "sratoolkit": "0"}}, {"Alternatives": null, "@attributes": {"cluster": "public", "filename": "KZ1354_GA9939_R1.fastq.gz", "size": "10546554601", "date": "2020-03-05 12:28:05", "md5": "cc710b24f968ded71c2f3ce78b342d1a", "version": "1", "semantic_name": "fastq", "supertype": "Original", "sratoolkit": "0"}}]