{"ID": "doi:10.7554/eLife.21856", "lab": {"@type": ["Lab", "Item"], "correspondence": [{"contact_email": "c3RldmVoQGZoY3JjLm9yZw==", "@id": "/users/bed386a9-7ee0-4ac0-ab50-741018edf928/", "display_title": "Steve Henikoff"}], "display_title": "Steve Henikoff, FREDHUTCH", "status": "current", "@id": "/labs/steve-henikoff-lab/", "uuid": "3a41e042-a9f3-4205-a28a-4c20ba2edda2", "title": "Steve Henikoff, FREDHUTCH", "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin", "role.lab_submitter", "submits_for.3a41e042-a9f3-4205-a28a-4c20ba2edda2"]}, "pi": {"error": "no view permissions"}}, "url": "https://www.ncbi.nlm.nih.gov/pubmed/28079019", "award": {"center_title": "TCPA - Henikoff", "description": "TCPA: The 4D Nucleome project focuses on describing the 3D organization within the nucleus, with \nthe ultimate goal of understanding this organization in mechanistic terms. Our project uses novel methods for epigenomic profiling that detect 3D contact sites without cross-linking and with much higher resolution than current technologies. We recently introduced a novel strategy for chromatin profiling called CUT&RUN (Cleavage Under Targets & Release Using Nuclease), in which antibody-targeted controlled cleavage by micrococcal nuclease releases specific protein-DNA complexes into the supernatant for paired-end DNA sequencing. The method yields precise transcription factor (TF) profiles, yet is simple to perform and is inherently robust, with extremely low backgrounds requiring ~1/10 th the sequencing depth of chromatin immunoprecipitation (ChIP). CUT&RUN binding and cleavage occurs in situ, allowing for both quantitative high-resolution chromatin mapping and probing of the 3D chromatin environment. Together with our new native ChIP-seq protocol, we distinguish direct \u201canchor\u201d sites of a \nchromatin-bound protein from contacting sites without fixation, a kind of inference has not been possible with current methods of interrogating 3D chromatin architecture. We have two Aims: First, we will annotate the genome at high density for CTCF and cohesin sites, distinguishing between anchor and contact sites in 4D Nucleome cell lines. Second, we will continue development of a new replacement technology for 3D contact mapping, using CUT&RUN as a \u201cfront-end\u201d for proximity ligation (CUT&PASTE \u2013 Cleave Under Targets & Polish And Splice Touching Ends), building on our novel sci-HiC protocol for high-resolution TF-specific 3D interaction mapping. By annotating more contact sites in genomes and assigning the directionality of contacts, we move towards a mechanistic model of how topology within the nucleus is organized. Participation in the 4DN program would provide the opportunity to compare and integrate our CUT&RUN/PASTE datasets with 4DN datasets on common cell lines and differentiating tissues, ideally positioning consortium investigators to adopt our alternative strategy for their own 4DN efforts.", "project": "4DN", "uuid": "4ddcac2d-72b6-40a7-ac68-67efba21a0d0", "status": "current", "name": "TCPA-2017-04", "display_title": "TETHERED NUCLEASE STRATEGIES FOR IN SITU MAPPING OF 3D NUCLEAR ORGANIZATION", "@id": "/awards/TCPA-2017-04/", "@type": ["Award", "Item"], "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin"]}, "pi": {"error": "no view permissions"}}, "title": "An efficient targeted nuclease strategy for high-resolution mapping of DNA binding sites.", "status": "current", "aliases": ["henikoff_lab:CUTnRUN_ref"], "authors": ["Skene PJ", "Henikoff S"], "journal": "eLife", "abstract": "We describe Cleavage Under Targets and Release Using Nuclease (CUT&RUN), a chromatin profiling strategy in which antibody-targeted controlled cleavage by micrococcal nuclease releases specific protein-DNA complexes into the supernatant for paired-end DNA sequencing. Unlike Chromatin Immunoprecipitation (ChIP), which fragments and solubilizes total chromatin, CUT&RUN is performed in situ, allowing for both quantitative high-resolution chromatin mapping and probing of the local  chromatin environment. When applied to yeast and human nuclei, CUT&RUN yielded precise transcription factor profiles while avoiding crosslinking and solubilization issues. CUT&RUN is simple to perform and is inherently robust, with extremely low backgrounds requiring only ~1/10th the sequencing depth as ChIP, making CUT&RUN especially cost-effective for transcription factor and chromatin profiling. When used in conjunction with native ChIP-seq and applied to human CTCF, CUT&RUN mapped directional long range contact sites at high resolution. We conclude that in situ mapping of protein-DNA interactions by CUT&RUN is an attractive alternative to ChIP-seq.", "date_created": "2018-02-23T08:37:56.547785+00:00", "submitted_by": {"error": "no view permissions"}, "date_published": "2017-01-16", "public_release": "2018-02-23", "schema_version": "2", "@id": "/publications/059e0fe2-eca8-441d-86c5-20e57d531b87/", "@type": ["Publication", "Item"], "uuid": "059e0fe2-eca8-441d-86c5-20e57d531b87", "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin"]}, "display_title": "Skene PJ and Henikoff S (2017) doi:10.7554/eLife.21856", "external_references": [], "short_attribution": "Skene PJ and Henikoff S (2017)", "@context": "/terms/", "aggregated-items": {}, "validation-errors": []}