EXPERIMENT_TITLE: GSM6042041: HiC_K562_shCTCFshRAD21_rep2; Homo sapiens; Hi-C
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: For Hi-TrAC and Hi-C, genomic DNA was purified by Phenol-Chloroform extraction. RNA was extracted with QIAzol lysis reagent (QIAGEN) and RNeasy mini kit (QIAGEN). For ChIP-seq and ATAC-seq, genomic DNA was purified with QIAGEN MinElute Reaction Cleanup Kit. For Hi-TrAC, repair DNA gaps with T4 DNA polymerase. Remove free bridging linker by AMPure XP beads. Then, Digest purified DNA with restriction enzymes MluCI/NlaIII, and enrich biotin-labeled DNA fragments with streptavidin beads. Perform adapter ligation on beads. After washing the beads, run PCR with illumina multiplexing indexed primers. For RNA-seq, libraries were constructed following Smart-seq2 protocol (Picelli et al., 2014). For ChIP-seq, DNA was purified, then end-repaired using End-It DNA End-Repair kit (Epicentre). A-tailing was performed with Klenow Fragment (3'->5' exo-) with the presence of dATP. Universal adapters were then ligated. The libraries were then amplified by PCR using indexed primers. For ATAC-seq, purified genomic DNA was amplified by PCR with indexed primers to construct the libraries. For Hi-C, purified genomic DNA was treated with T4 DNA polymerase to remove biotin labels at DNA ends. Fragment the DNA by sonication to 300-500 bp. Fragmentated DNA was end-repaired, followed with A-tailing. Biotin labeled DNA was enriched with MyOne Streptavidin C1 beads (Invitrogen). PCR adapters were then ligated to the enriched DNA on beads. Perform PCR with indexed primers to amplify the libraries.
STUDY_DESCRIPTOR_STUDY_ABSTRACT: The three-dimensional genomic structure plays a critical role in gene expression, cellular differentiation, and pathological conditions. Previous studies have extensively investigated the structures including A/B compartments and topologically associating domains (TADs) at a scale from hundreds of kilobases (kb) to megabases (Mb). However, fine-scale chromatin architectures, particularly enhancer-promoter interactions, which are often within 100 kb and critical for the temporospatial regulation of expression, remain to be fully characterized due to technical limitations. In this study, we report Hi-TrAC as a proximity ligation free, robust, and sensitive technique to profile genome-wide chromatin interactions at high-resolution among accessible regulatory elements. Hi-TrAC detects chromatin looping among accessible chromatin regions at single nucleosome resolution. We observed that cell-specifically expressed genes are harbored in active sub-TADs. With almost half million identified loops, we constructed a comprehensive interaction network of regulatory elements across the genome. After integrating chromatin binding profiles of transcription factors (TFs), we discovered that cohesin complex and CTCF are responsible for organizing long-range chromatin loops, which are related with domain formation, whereas ZNF143 and HCFC1 are involved in structuring short-range chromatin loops between regulatory elements, which directly regulate gene expression.  Thus, here we developed a new methodology to identify a delicate and comprehensive network of cis regulatory elements, revealing the complexity and a division of labor of TFs in chromatin looping for genome organization and gene expression. Overall design: The genome structure of GM12878, K562 and mESC cells was studied by a newly developed technique Hi-TrAC. Chromatin binding profiles of architectural proteins CTCF, RAD21, HCFC1 and ZNF143 were studied by ChIP-seq. The effect of these four proteins on gene expression was investigated by RNA-seq, accessbility changes were studied by ATAC-seq, changes of chromatin interactions were also validated by Hi-C.
SAMPLE_TITLE: HiC_K562_shCTCFshRAD21_rep2
SAMPLE_SAMPLE_ATTRIBUTES_SAMPLE_ATTRIBUTE: [{"TAG": "source_name", "VALUE": "K562"}, {"TAG": "cell line", "VALUE": "K562"}, {"TAG": "tissue", "VALUE": "K562"}, {"TAG": "genotype/variation", "VALUE": "CTCF RAD21 double knockdown"}]
Pool_Member_@attributes_sample_title: HiC_K562_shCTCFshRAD21_rep2
RUN_SET_RUN_Pool_Member_@attributes_sample_title: HiC_K562_shCTCFshRAD21_rep2
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