EXPERIMENT_TITLE: GSM2627219: RWPE1_HiC_rep1; Homo sapiens; Hi-C
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: RNA was prepared using TRIzol reagent (Thermal Fisher Scientific, cat. no. 15596018). 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. The RNA-seq libraries were made using KAPA stranded mRNA-seq kits (KAPK Biosystems, cat. no. kk8421) according to manufacturer's instructions 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: Prostate cancer (PCa) is the leading cancer among men in the United States, with genetic factors contributing to ~42% of the susceptibility to PCa. To understand the causal genetic factors associated with PCa, we have focused on a PCa risk region located at 7p15.2. We performed Hi-C analysis and demonstrated that this region has long range interactions with the HOXA locus, located ~873 kb away. Using the CRISPR/Cas9 system, we deleted a 4 kb region encompassing several prostate cancer risk-associated SNPs and performed RNA-seq to investigate transcriptomic changes in RWPE1 prostate cells lacking the regulatory element. Our results suggest that the risk element affects the expression of HOXA13 and HOTTIP, but not other genes in the HOXA locus, via a repressive loop. Forced expression of HOXA13 was also performed to gain insight into the mechanisms by which this risk element affects prostate cancer risk. Overall design: Examination of normal prostate epithelial cell RWPE-1 genomic interactions by Hi-C. A prostate cancer risk region at 7p15.2 was deleted and HOXA13 gene was forced expressed in RWPE-1 cells, the transcriptomic changes were investigated by RNA-seq.
SAMPLE_TITLE: RWPE1_HiC_rep1
SAMPLE_SAMPLE_ATTRIBUTES_SAMPLE_ATTRIBUTE: [{"TAG": "source_name", "VALUE": "RWPE1"}, {"TAG": "cell line", "VALUE": "prostate cell line RWPE1"}, {"TAG": "treatment", "VALUE": "None"}, {"TAG": "genotype/variation", "VALUE": "wild type"}]
Pool_Member_@attributes_sample_title: RWPE1_HiC_rep1
RUN_SET_RUN_Pool_Member_@attributes_sample_title: RWPE1_HiC_rep1
RUN_SET_RUN_SRAFiles_SRAFile: [{"Alternatives": null, "@attributes": {"cluster": "public", "filename": "RWPE1_HiC_rep1_R1.fastq.gz", "size": "21894435809", "date": "2020-04-14 22:32:58", "md5": "e2005545f2a88473492b4ca460e749cc", "version": "1", "semantic_name": "fastq", "supertype": "Original", "sratoolkit": "0"}}, {"Alternatives": null, "@attributes": {"cluster": "public", "filename": "RWPE1_HiC_rep1_R2.fastq.gz", "size": "23690625639", "date": "2020-04-14 22:32:58", "md5": "815c356d3f5e40fbcabd5335ed86fac0", "version": "1", "semantic_name": "fastq", "supertype": "Original", "sratoolkit": "0"}}, {"Alternatives": [null, null, null], "@attributes": {"cluster": "public", "filename": "SRR5558500.sralite", "url": "https://sra-downloadb.be-md.ncbi.nlm.nih.gov/sos5/sra-pub-zq-14/SRR005/558/SRR5558500.sralite.1", "size": "19042304529", "date": "2021-06-16 09:16:37", "md5": "a6e82cd43c4567e00360a04589479da0", "version": "1", "semantic_name": "SRA Lite", "supertype": "Primary ETL", "sratoolkit": "1"}}, {"Alternatives": null, "@attributes": {"cluster": "public", "filename": "SRR5558500", "url": "https://sra-pub-run-odp.s3.amazonaws.com/sra/SRR5558500/SRR5558500", "size": "31007343041", "date": "2017-05-15 11:40:23", "md5": "41e127b6ebc171301e7457ea933d925e", "version": "1", "semantic_name": "SRA Normalized", "supertype": "Primary ETL", "sratoolkit": "1"}}]