EXPERIMENT_TITLE: GSM3358193: RWPE1_HiC_rep1 (duplicate of GSM2627219); Homo sapiens; Hi-C
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: In situ Hi-C experiments were performed in RWPE-1 cells following the original protocol by Rao et al with minor modifications. Hi-C was performed in duplicate and 5 x 106 cells were used for each experiment. 100U of MboI restriction enzyme (NEB, R0147) was used to digest chromatin. For ligation, 2000U T4 DNA Ligase (NEB, M0202) was added and incubated at room temperature for 4 hours with slow rotation. Hi-C material was sheared to a size of 300-500bp using a Covaris instrument (Covaris S2, Woburn, MA). Biotin-tagged DNA was pulled down using Dynabeads MyOne Streptavidin C1 beads (Life technologies, 65002) with 2X Binding Buffer (2X BB: 10mM Tris-HCl (pH 7.5), 1nM EDTA, 2M NaCl). Hi-C library was amplified with 14 cycles of PCR using Illumina primers. Each library was sequenced with 75bp paired-end for ~500M read pairs using Illumina HiSeq 2000. In situ HiC experiments were performed in RWPE-1 cells following the original protocol by Rao et al with minor modifications. HiC was performed in duplicate and 5 x 106 cells were used for each experiment. 100U of MboI restriction enzyme (NEB, R0147) was used to digest chromatin. For ligation, 2000U T4 DNA Ligase (NEB, M0202) was added and incubated at room temperature for 4 hours with slow rotation. HiC material was sheared to a size of 300-500bp using a Covaris instrument (Covaris S2, Woburn, MA). Biotin-tagged DNA was pulled down using Dynabeads MyOne Streptavidin C1 beads (Life technologies, 65002) with 2X Binding Buffer (2X BB: 10mM Tris-HCl (pH 7.5), 1nM EDTA, 2M NaCl). HiC library was amplified with 14 cycles of PCR using Illumina primers.
STUDY_DESCRIPTOR_STUDY_ABSTRACT: Background: Recent genome-wide association studies (GWAS) have identified more than 100 loci associated with increased risk of prostate cancer, most of which are in non-coding regions of the genome. Understanding the function of these non-coding risk loci is critical to elucidate the genetic susceptibility to prostate cancer. Results: We generated genome-wide regulatory element maps and performed genome-wide chromosome confirmation capture assays (in situ Hi-C) in normal and tumorigenic prostate cells. Using this information, we annotated the regulatory potential of 2,181 fine-mapped PCa risk-associated SNPs and predicted a set of target genes that are regulated by PCa risk-related H3K27Ac-mediated loops. We next identified PCa risk-associated CTCF sites involved in long-range chromatin loops. We used CRISPR-mediated deletion to remove PCa risk-associated CTCF anchor regions and the CTCF anchor regions looped to the PCa risk-associated CTCF sites; we observed up to 100 fold increases in expression of genes within the loops when the PCa risk-associated CTCF anchor regions were deleted.  Conclusions: We have identified GWAS risk loci involved in long-range loops that function to repress gene expression within chromatin loops. Our studies provide new insights into the genetic susceptibility to prostate cancer. Overall design: ChIP-seq, RNA-seq, Hi-C in prostate cells.
SAMPLE_TITLE: RWPE1_HiC_rep1 (duplicate of GSM2627219)
SAMPLE_SAMPLE_ATTRIBUTES_SAMPLE_ATTRIBUTE: [{"TAG": "source_name", "VALUE": "prostate cell line RWPE1"}, {"TAG": "cell line", "VALUE": "RWPE1"}, {"TAG": "genotype", "VALUE": "wild type"}]
Pool_Member_@attributes_sample_title: RWPE1_HiC_rep1 (duplicate of GSM2627219)
RUN_SET_RUN_Pool_Member_@attributes_sample_title: RWPE1_HiC_rep1 (duplicate of GSM2627219)
RUN_SET_RUN_SRAFiles_SRAFile: [{"Alternatives": null, "@attributes": {"cluster": "public", "filename": "SRR7760386", "url": "https://sra-pub-run-odp.s3.amazonaws.com/sra/SRR7760386/SRR7760386", "size": "31007343073", "date": "2018-08-28 13:26:57", "md5": "44373c77aec3cc029fc664f314df8f85", "version": "1", "semantic_name": "SRA Normalized", "supertype": "Primary ETL", "sratoolkit": "1"}}, {"Alternatives": [null, null, null], "@attributes": {"cluster": "public", "filename": "SRR7760386.sralite", "url": "https://sra-downloadb.be-md.ncbi.nlm.nih.gov/sos5/sra-pub-zq-11/SRR007/760/SRR7760386/SRR7760386.sralite.1", "size": "19042304529", "date": "2020-06-21 13:15:01", "md5": "88a896caa87e43343d607aa3607d89a2", "version": "1", "semantic_name": "SRA Lite", "supertype": "Primary ETL", "sratoolkit": "1"}}]