EXPERIMENT_TITLE: GSM2665657: BCBL1_TREx-KRta_NoDox_CaptureHiC; Homo sapiens; Hi-C
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: Chromatin prepared from KSHV-infected cells for the 3C procedure described above was used as input for Capture Hi-C. Briefly, infected TREx-K-Rta BCBL-1 or iSLK/r.219 cells (1 x 10^8) were fixed with formaldehyde, and the chromatin was digested with either BamHI or DpnII restriction enzyme and re-ligated under ultra-dilute conditions. DNA-protein crosslinks were reversed by incubation at 65°C overnight with 0.2% SDS. The DNA was then purified with the QIAquick PCR Purification Kit (Qiagen). Capture Hi-C sequencing libraries enriched for KSHV genome re-ligation products were prepared from DNA samples obtained from chromosome conformation capture (3C) 3analysis of TREx-K-Rta BCBL-1 and iSLK.r219 cells. The DNA preparations were sheared to a mean fragment length of 250 bp using a Covaris E220 Focused-ultrasonicator (Covaris, Inc.) and sequencing libraries were then prepared from the DNA chimerae using the NEBNext DNA Library Prep Kit (New England BioLabs, Inc.). Target enrichment for KSHV genomic content was performed using a custom-designed KSHV genomic capture probe library and according to the manufacturer’s standard protocols (IDT, Coralville, Iowa). Briefly, in-solution hybrid capture was performed by hybridizing libraries (500 ng) with the KSHV genomic capture probe pool (3 pmol) in a mixture containing xGen 1X Hybridization Buffer, Cot-1 (5 µg), and xGen Universal Blocking Oligos at a temperature of 65°C for 4 hours. Hybridized targets were then bound to streptavidin-coupled magnetic beads (Dynabeads M-270 Streptavidin beads; Thermo Fisher Scientific) by incubation at 65°C for 45 minutes, and unbound DNA was removed by a series of high-stringency (65°C) and low-stringency (room temperature) washes. The KSHV genome-enriched Capture Hi-C library DNA was eluted and post-capture PCR enrichment performed with high-fidelity KAPA HiFi HotStart DNA Polymerase (Kapa Biosystems, Inc., Wilmington, MA) and purified with Agencourt AMPure XP magnetic beads. KSHV-enriched libraries were then validated with an Agilent 2100 Bioanalyzer, and quantitated with a Qubit fluorometer (Invitrogen) and by qPCR-based quantification (KAPA Library Quantification Kit).
STUDY_DESCRIPTOR_STUDY_ABSTRACT: The three-dimensional structure of chromatin via formation of genomic loops allows cellular RNA polymerase II to access distal promoters, thus it has a significant impact on the outcome of gene expression. The Kaposi's sarcoma-associated herpesvirus (KSHV) entire lytic transcriptional program of 80 plus genes is initiated by a single viral transactivator, K-Rta. Here we examined the relationship between the KSHV genomic structure and K-Rta recruitment sites with unbiased Capture Hi-C analyses. The studies showed that KSHV forms a unique genomic structure, which coordinates a domain-specific viral gene expression program via K-Rta recruitment, and identified a number of direct physical, long-range, and dynamic genomic interactions. Chromatin immunoprecipitation-sequencing analyses identified a limited number of K-Rta recruitment sites in the KSHV genome, including the K12 and PAN RNA promoters. KSHV engineered to harbor point mutations in the K-Rta-responsive elements (RE) at these promoters significantly attenuated not only the direct downstream gene, but also distal viral gene expression in a domain-specific manner. Despite the long distance between two RE sites in the linear orientation, efficient recruitment of K-Rta to either promoter required intact K-Rta REs at both promoters. Finally, inducible genomic loops generated during KSHV reactivation occurred preferentially at K-Rta recruitment sites. Mutation of a K-Rta RE inhibited formation of inducible genomic loops, gene expression of its destination of looping genes, and KSHV replication in de novo infected human gingival epithelial cells. These results suggest that the KSHV genome is programed to form both stable and inducible chromatin hubs for effective KSHV gene expression, and partly explains why K-Rta has such a dominant effect on KSHV lytic gene transcription. Overall design: The overall goal of these studies was to define the long-range chromatin interactions occurring within the KSHV genome as well as between the KSHV and human host cell genomes both during latency and viral reactivation. These were defined in an unbiased manner using Hi-C analysis, which is chromosome conformation capture (3C) combined with next-generation sequencing (NGS). A modified protocol, referred to as Capture Hi-C, was utilized to selectively enrich interactions involving KSHV by performing target enrichment with a custom-designed KSHV genomic capture probe library. Experiments were performed in two cell lines, TREx-(F3H3)-K-Rta BCBL-1 and iSLK.r219 cells, in which KSHV can be reactivated via doxyclyine-induced expression of K-Rta. Accordingly, Capture Hi-C sequencing libraries were prepared from replicate cultures of each cell line cultured in the absence (i.e., latency) or presence (i.e., reactivation) of doxycycline for 24 hours.
SAMPLE_TITLE: BCBL1_TREx-KRta_NoDox_CaptureHiC
SAMPLE_SAMPLE_ATTRIBUTES_SAMPLE_ATTRIBUTE: [{"TAG": "source_name", "VALUE": "TREx-(F3H3)-K-Rta BCBL-1 cells"}, {"TAG": "tissue", "VALUE": "human primary effusion lymphoma"}, {"TAG": "cell type", "VALUE": "KSHV-positive human B cell lymphoma cells"}, {"TAG": "passages", "VALUE": "5~10"}, {"TAG": "restriction enzyme for dna digestion", "VALUE": "BamHI"}, {"TAG": "capture library", "VALUE": "KSHV"}]
Pool_Member_@attributes_sample_title: BCBL1_TREx-KRta_NoDox_CaptureHiC
RUN_SET_RUN_Pool_Member_@attributes_sample_title: BCBL1_TREx-KRta_NoDox_CaptureHiC
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