EXPERIMENT_TITLE: GSM7235226: ARPE19 cells, Hi-C, normal control; Homo sapiens; Hi-C
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: 2×107 cells were fixed in 1% formaldehyde for 10 min at room temperature and quenched with glycine. Fixed cells were permeabilized in 1 ml lysis buffer (10 mM Tris-HCl pH 8, 10 mM NaCl, 0.2% NP-40 and protease inhibitor cocktail (Sigma)) on ice for 15 min and spun down at 2,500 g for 5 min at 4℃. The pellet was resuspended in 345 μl of 1×NEBuffer r2.1 (NEB), and incubated with 38 μl of 1% SDS at 65℃ for 10 min. Next, 43 μl of 10% Triton X-100 was added to quench the SDS at 37℃ for 15 min. Then 12 μl of 10×NEBuffer r2.1 and 400 U HindIII (NEB) were added per aliquot, and mixed gently to digest the chromatin overnight at 37℃ on a rocking platform. On the next day, the HindIII restriction enzyme was inactivated at 65℃ for 25 min. Then, biotin-14-dATP (Life Technologies), dCTP, dGTP, dTTP and DNA polymerase I Klenow were added (NEB), and incubated at 37℃ for 2 h. The digested chromatin was diluted and re-ligated by T4 DNA ligase (NEB), incubated at 16℃ for 4 h, and shaken manually for three times. The chromatin products were de-cross-linked overnight by adding 50 μl of 10 mg ml-1 proteinase K (Sigma), and incubated at 65℃ overnight. DNA was extracted by phenol-chloroform extraction protocol. RNA was digested by adding 1 μl of RNase A (Sigma) and incubating at 37℃ for 30 min. Then T4 DNA polymerase (NEB) was used to remove biotin from un-ligation ends for 4 h at 20℃, and the enzymes were inactivated for 20 min at 75℃. DNA was purified and sheared to a size of 350 bp (Covaris M220). DNA size selection was conducted using AMPure XP beads (Beckman Coulter). Biotin-marked ligation products was isolated with MyOne Streptavidin C1 beads (Life Technologies). End repair, A adding, adaptor adding reaction, PCR amplification and DNA products size selection were performed sequentially. The libraries were sequenced by the Illumina PE150 paired-end sequencing platform.
STUDY_DESCRIPTOR_STUDY_ABSTRACT: TAD (Topologically Associating Domain) reorganization occurs commonly in cell nucleus and contributes to gene activation and inhibition through separation or fusion of adjacent TADs. However, identification of functional genes impacted by TAD alteration and revealing of TAD-reorganization mechanism underlying gene transcription remain to be fully elucidated. Here, we firstly developed a novel approach termed Inter3D to specifically dig out genes regulated by TAD reorganization. By constructing the comprehensive TAD reorganization medicating epigenomic landscapes in tumor cells, we showed that TAD separation interrupted the intrachromosomal loop at MYL12B locus and abrogated its transcription, while TAD fusion generated the chromosomal interaction and triggered CYP27B1 activation. Our study provides a comprehensive insight into capture of TAD rearrangement-mediated gene loci and moves towards recognizing the functional role of TAD reorganization in gene transcription. Overall design: To address the questions about TAD reorganization, we developed a novel easy-to-use pipeline for Interactome identification with 3D multi-genomics (Inter3D) by performing integrative analysis of multi-omics datasets, including 3D genomics, epigenomics and transcriptomics. TAD boundaries are rich in various architectural proteins, among which we paid more attention to CTCF regulated ones. In details, Inter3D first identifies the CTCF-medicated TAD boundary alterations using Hi-C and CTCF ChIP-seq datasets of normal ARPE19 cells and cancer WERI-RB1 cells. Next, incorporating with the RNA-seq datasets of ARPE19 and WERI-RB1 cell lines, Inter3D identifies the candidate DEGs between variant samples regulated by CTCF binding alteration and TAD alteration. Finally, Inter3D identifies the candidate enhancers in promoter-distal peaks robust in ATAC-seq datasets of ARPE19 and WERI-RB1 cell lines, ruling out false positive target genes regulated by enhancer only rather than 3D structure.
SAMPLE_TITLE: ARPE19 cells, Hi-C, normal control
SAMPLE_SAMPLE_ATTRIBUTES_SAMPLE_ATTRIBUTE: [{"TAG": "source_name", "VALUE": "retinal pigment epithelia"}, {"TAG": "tissue", "VALUE": "retinal pigment epithelia"}, {"TAG": "cell line", "VALUE": "ARPE19"}, {"TAG": "cell type", "VALUE": "a spontaneously arising retinal pigment epithelia (RPE) cell line derived from the normal eyes of a 19-year-old male who died from head trauma in a motor vehicle accident"}, {"TAG": "genotype", "VALUE": "WT"}, {"TAG": "geo_loc_name", "VALUE": "missing"}, {"TAG": "collection_date", "VALUE": "missing"}]
RUN_SET_RUN_SRAFiles_SRAFile: [{"Alternatives": [null, null], "@attributes": {"cluster": "public", "filename": "A_2.clean.fq.gz", "url": "https://sra-pub-src-1.s3.amazonaws.com/SRR24327436/A_2.clean.fq.gz.1", "size": "93257229165", "date": "2023-04-27 21:37:28", "md5": "852168586168db48275b29edf34eb44f", "version": "1", "semantic_name": "fastq", "supertype": "Original", "sratoolkit": "0"}}, {"Alternatives": [null, null], "@attributes": {"cluster": "public", "filename": "A_1.clean.fq.gz", "url": "https://sra-pub-src-1.s3.amazonaws.com/SRR24327436/A_1.clean.fq.gz.1", "size": "83456915412", "date": "2023-04-27 21:34:53", "md5": "e31bbe364087628a484e09d15c44f173", "version": "1", "semantic_name": "fastq", "supertype": "Original", "sratoolkit": "0"}}]