EXPERIMENT_TITLE: GSM7923253: RORA_OE_capture_hic_2; Homo sapiens; Hi-C
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: The chromatin DNA was digested with 100 U HindIII (NEB, #R3104S) and labeled with biotin-14-dCTP (Invitrogen, #19518018). The biotin-labeled DNA fragments were then ligated by 50 U T4 DNA ligase (NEB, # M0202S), de-crosslinked and extracted using QIAamp DNA Mini Kit (Qiagen). We then sheared the purified DNA fragments to 300-600 bp, repaired the ends and added A-tails and SureSelect adaptors. After biotin-streptavidin-mediated pull-down, DNA fragments were amplified by PCR. We conducted capture Hi-C of promoters by the SureSelect XT Library Prep Kit ILM (Agilent Technologies, #G9981A), as per the manufacturer's instructions, with custom-designed biotinylated RNA bait library and custom paired-end blockers. After another PCR amplification, promoter capture Hi-C libraries were purified with AMPure XP beads (Beckman Coulter, #a63880) Promoter capture Hi-C-seq
STUDY_DESCRIPTOR_STUDY_ABSTRACT: Proper differentiation of corneal epithelial cells (CECs) from limbal stem/progenitor cells (LSCs) is required for maintaining ocular homeostasis and clear vision. Here, using a single-cell transcriptomic atlas, we delineate the comprehensive and refined molecular regulatory dynamics during human CEC development and differentiation. We find that RORA is a CEC-specific molecular switch that initiates and drives LSCs to differentiate into mature CECs by activating PITX1. RORA dictates CEC differentiation by establishing CEC-specific enhancers and chromatin interactions between CEC gene promoters and distal regulatory elements. Conversely, RORA silences LSC-specific promoters and disrupts promoter-anchored chromatin loops to turn off LSC genes. Collectively, our work provides detailed and comprehensive insights into the transcriptional dynamics and RORA-mediated epigenetic remodeling underlying human corneal epithelial differentiation. Overall design: Promoter capture Hi-C-seq in control and RORA-overexpressed LSCs.
SAMPLE_TITLE: RORA_OE_capture_hic_2
SAMPLE_SAMPLE_ATTRIBUTES_SAMPLE_ATTRIBUTE: [{"TAG": "source_name", "VALUE": "limbal stem cells"}, {"TAG": "cell type", "VALUE": "primary limbal stem cells"}, {"TAG": "genotype", "VALUE": "RORA overexpression"}, {"TAG": "geo_loc_name", "VALUE": "missing"}, {"TAG": "collection_date", "VALUE": "missing"}]
Pool_Member_@attributes_sample_title: RORA_OE_capture_hic_2
RUN_SET_RUN_Pool_Member_@attributes_sample_title: RORA_OE_capture_hic_2
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