EXPERIMENT_TITLE: GSM3489420: 19: HiC_S1; Homo sapiens; Hi-C
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: Following treatment cells were fixed with Formaldehyde for Hi-C. Nuclei were obtained after lysis on ice for 30 minutes with 10 mM Tris-HCl pH8, 10 mM NaCl, 0.2% Igepal CA-630 and 1 Roche Complete EDTA-free tablet. After 10 minutes treatment with 0.1% SDS at 37°C, nuclei were incubated with Triton 10% for 10 minutes also at 37°C. Nuclei were then suspended in 1X DpnII buffer (NEB) and chromatin digested overnight with 400U of DpnII (NEB) at 37°C. Following an additional 4-hour incubation with extra 400U of DpnII, digestion was stopped by 65°C incubation for 30 minutes. DNA ends were then filled in with dATP-Biotin using polymerase klenow for 60 minutes at 37°C. Ligation was performed in-situ overnight (with nuclei intact) using 2000U of T4 DNA ligase from NEB (M0202) at 16°C. Decrosslinking was performed overnight with Proteinase K at 65°C. 40 mg of unligated biotin ends were removed by incubation with T4 DNA polymerase for 4 hours at 20°C. DNA fragments were then sonicated using an LE Covaris 220 instrument using 450 PIP, 30% duty factor, 200 cycles per burst for 60 seconds. Following sonication, DNA fragments were end-repaired using T4 DNA polymerase, T4 PNK and Klenow polymerase (all from NEB). DNA fragments were then bound to Streptavidin C1 beads and washed following manufacturer's instructions and using 150 ml per 2.5 mg of sonicated DNA. A-tailing and adaptor ligation was then performed with DNA fragments bound to beads. A-tailing was done using Klenow polymerase without 3' exonuclease activity. For ligation, adaptors from the NEBNext DNA library prep reagent set (E6040) were used and ligated overnight. Following ligation, enzyme USER treatment was used to open the stem loop. Hi-C library amplification was then amplified using 7 PCR cycles, with Phusion polymerase and PCR primers from NEBNext Multiplex Oligos for Illumina (E7335) and resulted in approximately 1 mg of Hi-C material.
STUDY_DESCRIPTOR_STUDY_ABSTRACT: The organization of chromatin in the nucleus plays an essential role in gene regulation. About half of the mammalian genome comprises transposable elements. Given their repetitive nature, reads associated with these elements are generally discarded or randomly distributed among elements of the same type in genome-wide analyses. Thus, it is challenging to identify the activities and properties of individual transposons. As a result, we only have a partial understanding of how transposons contribute to chromatin folding and how they impact gene regulation. Using adapted PCR and Capture-based chromosome conformation capture (3C) approaches, collectively called 4Tran, we take advantage of the repetitive nature of transposons to capture interactions from multiple copies of endogenous retrovirus (ERVs) in the human and mouse genomes. With 4Tran-PCR, reads are selectively mapped to unique regions in the genome. This enables the identification of transposable element interaction profiles for individual ERV families and integration events specific to particular genomes. With this approach, we demonstrate that transposons engage in long-range intra-chromosomal interactions guided by the separation of chromosomes into A and B compartments as well as topologically associated domains (TADs). In contrast to 4Tran-PCR, Capture-4Tran can uniquely identify both ends of an interaction that involve retroviral repeat sequences, providing a powerful tool for uncovering the individual transposable element insertions that interact with and potentially regulate target genes. 4Tran provides new insight into the manner in which transposons contribute to chromosome architecture and identifies target genes that transposable elements can potentially control. Overall design: We used the Hi-C protocol to identify chromosomal interactions from HeLa cells treated with interferon gamma.
SAMPLE_TITLE: 19: HiC_S1
SAMPLE_SAMPLE_ATTRIBUTES_SAMPLE_ATTRIBUTE: [{"TAG": "source_name", "VALUE": "HeLa"}, {"TAG": "tissue", "VALUE": "HeLa F2 cells"}]
Pool_Member_@attributes_sample_title: 19: HiC_S1
RUN_SET_RUN_Pool_Member_@attributes_sample_title: 19: HiC_S1
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