EXPERIMENT_TITLE: GSM3564254: in situ Hi-C in C42B rep1; Homo sapiens; Hi-C
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: In situ Hi-C experiments were performed following the original protocol by Rao et al with minor modifications. In situ 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). ChIP assays were performed in C42B, 22Rv1 and RWPE1 cells using H3K9me3 (Cat# 13969 Lot# 1, Cell Signaling and Technology, Inc.), H3K27me3 (Cat# 9733 Lot# 8, Cell Signaling and Technology, Inc.), and H3K36me3 (Cat# 2901 Lot# 3, Cell Signaling and Technology, Inc.) antibodies, according to ENCODE standards (https://www.encodeproject.org/data-standards/). RNA-seq was performed in triplicate for RWPE1, C42B, and 22Rv1 cells.  RNA was extracted using Trizol reagent (Cat # 15596-018, Thermo Fisher Scientific, NY, USA) and the quality of RNA was assessed using a 2100 Bioanalyzer instrument (Cat # G2939AA, Agilent technologies), as described previously. For NOMe-seq, after isolating nuclei from the cells, M.CviPI was treated to methylate accessible GpCs. After purifying M.CvPI-treated DNA, sonication was performed. Bisulfite treatment of M.CviPI-methylated DNA resulted to convert all unmethylated Cs to Ts. In situ Hi-C ibraries were prepared using Kapa Hyper prep kit (Kapa #KK8503) according to the provided protocol. Library was amplified with PCR using Illumina primers. ChIP-seq: Libraries were barcoded (NEXTflex™ DNA Barcodes) (Bio Scientific, Austin, TX). RNA-seq libraries were made using KAPA Stranded mRNA-Seq Kit with KAPA mRNA Capture Beads (KK8420) (Kapa Biosystems, Woburn, MA). NOMe-seq: Libraries were generated using the Accel-NGS Methyl-Seq DNA Library Kit for Illumina Platforms.
STUDY_DESCRIPTOR_STUDY_ABSTRACT: A high-resolution 3D epigenomic map reveals insights into the creation of the prostate cancer transcriptome Prostate cancer (PCa) is the leading cancer among men in the United States. To understand gene regulation in 3D, chromatin interactions in prostate cancer cell is measured using in situ Hi-C. To better understand the impact of chromatin structure on regulation of the prostate cancer transcriptome, we developed high-resolution chromatin interaction maps in normal and prostate cancer cells using in situ Hi-C. By combining the in situ Hi-C data with active and repressive histone marks, CTCF binding sites, nucleosome-depleted regions, and transcriptome profiling, we identified topologically associating domains that changed in size and epigenetic states between normal and prostate cancer cells. Moreover, we identified normal and prostate cancer-specific enhancer-promoter loops and involved transcription factors. This creation of 3D epigenomic maps will enable a better understanding of prostate cancer biology and mechanisms of gene regulation. Overall design: Examination of prostate cancer epithelial cell 22Rv1 chromatin interactions using Hi-C In situ Hi-C, ChIP-seq using active and repressive histone marks and CTCF binding sites, NOMe-seq for nucleosome-depleted regions, and RNA-seq for transcriptome profiling in prostate epithelial cells to characterize prostate cancer epigenomic transcriptome. Please note that processed data was generated from all replicates and is linked to the corresponding rep1 sample records (or to Series records).
SAMPLE_TITLE: in situ Hi-C in C42B rep1
SAMPLE_SAMPLE_ATTRIBUTES_SAMPLE_ATTRIBUTE: [{"TAG": "source_name", "VALUE": "in situ Hi-C in C42B"}, {"TAG": "cell line", "VALUE": "C42B"}, {"TAG": "cell type", "VALUE": "Human prostate cancer cell line"}]
Pool_Member_@attributes_sample_title: in situ Hi-C in C42B rep1
RUN_SET_RUN_Pool_Member_@attributes_sample_title: in situ Hi-C in C42B rep1
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