EXPERIMENT_TITLE: GSM5032475: WT_D5_dEN_Rep2; Homo sapiens; Hi-C
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: cHi-C libraries were prepared from wild type or homozygous SOX17∆5'CTCF iPSC or dEN cells. Undifferentiated or day 5 differentiated ZIP13K224 cells were grown to a final count of 4-5 million, treated with Accutase (Sigma-Aldrich, A6964), resuspended and washed in DPBS. Cell lysis, NlaIII (NEB R0125) digestion, ligation and de-crosslinking was performed according to the Franke et al. protocol25. Adaptors were added to DNA and amplified according to Agilent instructions for Illumina sequencing. The library was hybridized to the custom-designed sure-select beads and indexed for sequencing of 200x106 fragments per sample (100 bp paired-end) following the Agilent instructions. Capture Hi-C experiments were performed as biological duplicates.
STUDY_DESCRIPTOR_STUDY_ABSTRACT: Recent investigation of insulated chromosome structures suggests their crucial role in controlling proper gene expression. However, it remains unclear whether developmental regulators can be precisely controlled by such insulated structures and whether developmental failure is caused by disrupting the structure boundaries. Here we identified topologically insulated regions (TIRs) from deeply sequenced human embryonic stem cell (hESC) Hi-C data and analyzed the gene distribution across TIRs. We find a notable enrichment of developmental regulators isolated from other genes by TIRs and show that the boundary CTCF sites are highly conserved across species. Perturbation of such boundaries in hESCs leads to failure to properly differentiate. Enhancer activity is restricted by such boundaries, further pointing towards their regulatory importance. Finally, germline mutations affecting such boundaries are subject to purifying selection and are underrepresented in the human population. Our findings provide new insights into mammalian genome organization and suggest a possible role of gene insulation through chromosomal folding structures for precise control of developmental regulators. Overall design: Capture HiC-Seq of SOX17 upstream CTCF wildtype and knockout in embryonic differentiation
SAMPLE_TITLE: WT_D5_dEN_Rep2
SAMPLE_SAMPLE_ATTRIBUTES_SAMPLE_ATTRIBUTE: [{"TAG": "source_name", "VALUE": "Human induced pluripotent stem cell (hiPSC)"}, {"TAG": "cell line", "VALUE": "ZIP13K2"}, {"TAG": "perturbation", "VALUE": "Wildtype"}, {"TAG": "differentiation days", "VALUE": "day 5"}]
Pool_Member_@attributes_sample_title: WT_D5_dEN_Rep2
RUN_SET_RUN_Pool_Member_@attributes_sample_title: WT_D5_dEN_Rep2
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