EXPERIMENT_TITLE: GSM3731627: HuES64_dEN_HIC2647_MboI; Homo sapiens; Hi-C
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: Hi-C libraries were prepared following the protocol described in Rao et. al. 20141. Briefly, one million cells were crosslinked with 1% formaldehyde for 10  minutes at room temperature and then quenched with 0.2M glycine solution. Cells were lysed and nuclei permeabilized with 0.5% sodium dodecyl sulphate for 10 minutes at 62°C. Chromatin was digested with 100 U of MboI restriction enzyme (New England Biolabs). Ends of the restriction fragments were blunted and labeled with a biotinylated nucleotide and then ligated. Nuclei were pelleted, proteins were digested with proteinase K and crosslinks were reversed by heating at 68°C overnight. DNA was sheared in a Covaris focused ultrasonicator to average fragment length of 400 bp. Size-selected DNA was enriched for biotinylated ligation products through binding to T1 streptavidin beads (Thermo Fisher). Libraries were prepared for Illumina sequencing by performing the end-repair, A-tailing and adapter ligation steps with DNA attached to the beads. Hi-C libraries were amplified directly off the beads and purified for subsequent Illumina sequencing with 100 paired-ends.
STUDY_DESCRIPTOR_STUDY_ABSTRACT: Developmental regulators are spatiotemporal regulated in the process of human development. Recent investigation of insulated chromosome structures suggests their crucial role in control of proper gene expression. However, it's unclear whether developmental regulators can be precisely controlled by such insulated structures and whether developmental failure can be 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 found 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 the differentiation failure. Germline mutations affecting such boundaries are subject to purifying selection, and are underrepresented in the human population. Further analyses demonstrated the enhancer activity is restricted by such boundaries that further points towards regulatory importance. Our findings provide new insights into mammalian genome organization and including a possible role of gene insulation through chromosomal folding structures for precise control of developmental regulators. Overall design: HiC-Seq of 4 cell lines
SAMPLE_TITLE: HuES64_dEN_HIC2647_MboI
SAMPLE_SAMPLE_ATTRIBUTES_SAMPLE_ATTRIBUTE: [{"TAG": "source_name", "VALUE": "Human embryonic stem cell (hESC)"}, {"TAG": "cell type", "VALUE": "endoderm"}, {"TAG": "restriction enzyme", "VALUE": "MboI"}]
Pool_Member_@attributes_sample_title: HuES64_dEN_HIC2647_MboI
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