EXPERIMENT_TITLE: GSM5753534: LSC Hi-C rep1; Homo sapiens; Hi-C
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: For ChIP-seq, chromatin was fixed with 1% formaldehyde for 10 min and sheared in a sonification buffer. Fragmented DNA was incubated with primary antibodies overnight at 4℃ and then with Protein A/G Dynabeads  for 1 h. The beads were collected and washed sequentially with the following solutions: high-salt buffer, low-salt wash buffer, and TE buffer.  Elution of the immunoprecipitated protein/DNA complexes was performed using elution buffer at 65℃ for 4 h. Next, the protein/DNA complexes were incubated with proteinase K (Invitrogen) and RNase A (Invitrogen) for 1 h. De-crosslinked DNA was purified using the PCR Purification Kit (Qiagen). For Hi-C, Following cellular cross-linking with 1% formaldehyde and nuclei extraction in hypotonic solution, the genomic DNA was digested with restriction enzyme MboI (NEB) at 37 °C overnight and labelled with biotinylated nucleotides when the cohesive ends were filled. Then, blunt-end proximity ligation was performed with T4 DNA ligase (NEB) at 16 °C for 4 h. After decrosslinking with proteinase K (Thermo) at 65°C overnight, ligated DNA was purified by QIAamp DNA Mini Kit (Qiagen) and sheared to ~400 bp. Next, Dynabeads™ MyOne™ Streptavidin C1 (Invitrogen) was used to pull down the biotin-marked ligation junctions. Purified DNA was used to prepare ChIP-seq DNA libraries for Illumina sequencing by KAPA Hyper Prep Kit (KAPABIOSYSTEMS) according to the manufacturer's instructions. The Hi-C library was generated using NEBNext Ultra™ II DNA library Prep Kit (NEB) according to the manufacturer's instructions. ChIP-seq, Hi-C
STUDY_DESCRIPTOR_STUDY_ABSTRACT: The insights into how genome topology couples with epigenetic states to govern the function and identity of the corneal epithelium are poorly understood. Here, we generate a high-resolution Hi-C interaction map of human limbal stem/progenitor cells (LSCs) and show that chromatin multi-hierarchical organisation is coupled to gene expression. By integrating Hi-C, epigenome and transcriptome data, we characterize the comprehensive 3D epigenomic landscapes of LSCs. We found that super-silencers mediate gene repression associated with corneal development, differentiation and disease via chromatin looping and/or proximity. Super-enhancer (SE) interaction analysis identified a set of SE interactive hubs that contribute to LSC-specific gene activation. These active and inactive element-anchored loop networks occur within the cohesin-occupied CTCF-CTCF loops. We further reveal a coordinated regulatory network of core transcription factors based on SE-promoter interactions. Our results provide detailed insights into the genome organization principle for epigenetic regulation of gene expression in stratified epithelia. Overall design: ChIP-seq for H3K9me2, CTCF, SMC1, p63 and EP300 in human limbal stem cells . Hi-C in human limbal stem cells.
SAMPLE_TITLE: LSC Hi-C rep1
SAMPLE_SAMPLE_ATTRIBUTES_SAMPLE_ATTRIBUTE: [{"TAG": "source_name", "VALUE": "limbal stem cells"}, {"TAG": "cell type", "VALUE": "primary limbal stem cells"}, {"TAG": "treatment", "VALUE": "untreated"}]
Pool_Member_@attributes_sample_title: LSC Hi-C rep1
RUN_SET_RUN_Pool_Member_@attributes_sample_title: LSC Hi-C rep1
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