EXPERIMENT_TITLE: GSM2588816: K562_R2; Homo sapiens; Hi-C
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: Hi-C 2.0 : We performed an updated protocol for Hi-C (Hi-C 2.0) that integrates recent improvements into a single protocol for efficient and high-resolution capture of chromatin interactions. One adaptation to the base protocol is the removal of a SDS solubilization step after digestion to better preserve nuclear structure. This prevents random ligation between released chromatin fragments by ligation in situ, i.e. in intact nuclei as previously described by (Rao SS,  et al .A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping. Cell 159, 2014, 1665–1680). A second adaptation in recently developed protocols increases the resolution of Hi-C through the use of restriction enzymes that digest more frequently, such as DpnII, or also described by Rao  et al .Thirdly, experimental steps can be included to reduce the number of uninformative sequences such as unligated (“dangling”) ends as described (Lieberman-Aiden, E. et al. Comprehensive Mapping of Long-Range Interactions Reveals Folding Principles of the Human Genome. Science 326, 289–293 (2009). Briefly, Cells were lysed and chromatin was digested with DpnII, then digested ends were filled in with biotinylated dATP and ligated in situ.  After reversing the formaldehyde crosslinks by incubation at 65 C with Proteinase K overnight, unligated biotinylated ends were removed by incubation with T4 DNA polymerase. The ligation products were fragmented by Covaris sonication and then size selected by Ampure fractionation.  The DNA ends were prepared for Illumina sequencing adapter ligation by repairing the DNA ends and adding an ‘A’ to each end.  The biotinylated junctions were then pulled down using MyOne streptavidin beads. Illumina paired end adapters were ligated onto the DNA ends and then the fragments were PCR amplified.   Samples were sequenced on an Illumina HiSeq 4000 using either a PE50 or PE100 bp read (reads were truncated to 50bp during mapping).
STUDY_DESCRIPTOR_STUDY_ABSTRACT: Chromosome conformation capture-based methods such as Hi-C have become mainstream techniques for the study of the 3D organization of genomes. These methods convert chromatin interactions reflecting topological chromatin structures into digital information (counts of pair-wise interactions).  Here, we describe an updated protocol for Hi-C (Hi-C 2.0) that integrates recent improvements into a single protocol for efficient and high-resolution capture of chromatin interactions. This protocol combines chromatin digestion and frequently cutting enzymes to obtain kilobase (Kb) resolution. It also includes steps to reduce random ligation and the generation of uninformative molecules, such as unligated ends, to improve the amount of valid intra-chromosomal read pairs. This protocol allows for obtaining information on conformational structures such as compartment and topologically associating domains, as well as high-resolution conformational features such as DNA loops. Overall design: HI-C 2.0
SAMPLE_TITLE: K562_R2
SAMPLE_SAMPLE_ATTRIBUTES_SAMPLE_ATTRIBUTE: [{"TAG": "source_name", "VALUE": "K562 cells"}, {"TAG": "cell line", "VALUE": "K562"}, {"TAG": "cell type", "VALUE": "lymphoblast"}, {"TAG": "Sex", "VALUE": "female"}]
Pool_Member_@attributes_sample_title: K562_R2
RUN_SET_RUN_Pool_Member_@attributes_sample_title: K562_R2
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