{"lab": {"uuid": "00073229-0955-47af-ab67-6abf4880cb2c", "@type": ["Lab", "Item"], "status": "current", "display_title": "Gerd Blobel, CHOP", "@id": "/labs/gerd-blobel-lab/", "title": "Gerd Blobel, CHOP", "correspondence": [{"contact_email": "YmxvYmVsQGVtYWlsLmNob3AuZWR1", "@id": "/users/9dcbdd0c-634c-4ec6-b6dc-89efd542c203/", "display_title": "Gerd Blobel"}], "pi": {"error": "no view permissions"}, "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin", "role.lab_submitter", "submits_for.00073229-0955-47af-ab67-6abf4880cb2c"]}}, "award": {"display_title": "ENGINEERING AND VISUALIZING GENOME FOLDING AT HIGH SPATIOTEMPORAL RESOLUTION", "@id": "/awards/1U01HL129998-01/", "status": "current", "name": "1U01HL129998-01", "center_title": "NT - Raj", "@type": ["Award", "Item"], "project": "4DN", "description": "NT: Critical unanswered questions in the field of genome biology are how the dynamics of chromatin folding shape gene expression patterns. Our knowledge of the dynamics of higher-order 3-D folding of chromatin is severely limited, largely due to the lack of technologies to precisely image, engineer and monitor looping in a precise spatiotemporal manner across a population of cells. Here we propose to address these limitations by developing tools to dynamically alter chromatin folding in a synchronous manner across populations of cells as well as individual cells, and measure chromatin looping and its relationship to transcription at high spatial resolution in single cells. In Specific Aim 1 we will design tools to control looping dynamics. We will modify factors that fold chromatin at various levels, such as Ldb1 and CTCF by fusion to a moiety whose stability can be controlled by diffusible ligands. In combination with hi resolution 5C and single molecule imaging these tools are expected to generate fundamental insights into the relationship of nuclear architecture and gene expression mechanisms. In Specific Aim 2 we plan to engineer light-inducible systems for the precise control of looping dynamics. Using light activated dimerization domains that can be used in conjunction with designer DNA binding proteins we attempt to engineer factors used to rapidly promote or disrupt chromatin looping at various scales. This technology should enable studies not only in populations but also at the single cell level. In Specific Aim 3: we will develp reagents to study the transcriptional dynamics in relation to looping at the single cell level. We will combine RNA FISH with super-resolution imaging to develop a methodology for exploring the spatial and temporal structure of nascent transcription at high resolution. Combined with high-throughput image acquisition, we will discriminate the temporal dynamics of transcription by measuring the relative intensities arising from the different parts of the transcript. We will employ super-resolution imaging (STORM) to measure the spatial structure of transcription sites. These experiments are expected to reveal the impact of forced chromatin looping on distinct stages of the transcription cycle and elucidate the relationship between transcriptional burst kinetics and physical gene structure.", "uuid": "cf0c686c-a3ad-411d-9986-ad42927352c0", "pi": {"error": "no view permissions"}, "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin"]}}, "study": "Cell Cycle", "status": "released", "aliases": ["blobel-lab:HiC-midG1"], "accession": "4DNESWLWNWV8", "condition": "mid-G1", "description": "2 biological replicates of Hi-C on G1E-ER4 cells in mid-G1 phase", "study_group": "Time Course", "date_created": "2020-03-03T20:07:45.028970+00:00", "submitted_by": {"error": "no view permissions"}, "dataset_label": "Hi-C on sync.G1E-ER4", "last_modified": {"modified_by": {"error": "no view permissions"}, "date_modified": "2021-01-25T04:58:03.633865+00:00"}, "public_release": "2020-04-15", "replicate_exps": [{"bio_rep_no": 1, "tec_rep_no": 1, "replicate_exp": {"accession": "4DNEXGDR6FZF", "display_title": "in situ Hi-C on G1E-ER4 with mCherry-MD with DpnII - 4DNEXGDR6FZF", "@type": ["ExperimentHiC", "Experiment", "Item"], "@id": "/experiments-hi-c/4DNEXGDR6FZF/", "uuid": "4c55ebe3-884e-4ce6-80e5-400ae9c3adca", "status": "released", "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin"]}}}, {"bio_rep_no": 2, "tec_rep_no": 1, "replicate_exp": {"accession": "4DNEX9K9WDJX", "display_title": "in situ Hi-C on G1E-ER4 with mCherry-MD with DpnII - 4DNEX9K9WDJX", "@type": ["ExperimentHiC", "Experiment", "Item"], "@id": "/experiments-hi-c/4DNEX9K9WDJX/", "uuid": "cdec6fa6-81f6-4dd2-9878-c26ea42421bd", "status": "released", "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin"]}}}], "schema_version": "2", "static_content": [{"content": {"name": "1ec3274f-4ad5-4383-a682-f1a91896fa44", "lab": {"display_title": "Gerd Blobel, CHOP", "uuid": "00073229-0955-47af-ab67-6abf4880cb2c", "@type": ["Lab", "Item"], "status": "current", "@id": "/labs/gerd-blobel-lab/", "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin", "role.lab_submitter", "submits_for.00073229-0955-47af-ab67-6abf4880cb2c"]}}, "@type": ["HiglassViewConfig", "UserContent", "Item"], "title": "4DNESWLWNWV8 - Processed files", "status": "released", "uuid": "1ec3274f-4ad5-4383-a682-f1a91896fa44", "display_title": "4DNESWLWNWV8 - Processed files", "award": {"display_title": "ENGINEERING AND VISUALIZING GENOME FOLDING AT HIGH SPATIOTEMPORAL RESOLUTION", "status": "current", "@id": "/awards/1U01HL129998-01/", "@type": ["Award", "Item"], "uuid": "cf0c686c-a3ad-411d-9986-ad42927352c0", "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin"]}}, "@id": "/higlass-view-configs/1ec3274f-4ad5-4383-a682-f1a91896fa44/", "description": "4DNESWLWNWV8 (2 biological replicates of Hi-C on G1E-ER4 cells in mid-G1 phase): 4DNFIQM3SAO3", "filetype": "HiglassViewConfig", "contributing_labs": [{"uuid": "b18699f9-e3e9-44e2-8070-d5b044efc09e", "@type": ["Lab", "Item"], "display_title": "Jennifer Cremins, UPENN", "@id": "/labs/jennifer-cremins-lab/", "status": "current", "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin", "role.lab_submitter", "submits_for.b18699f9-e3e9-44e2-8070-d5b044efc09e"]}}], "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin", "role.owner", "userid.7677f8a8-79d2-4cff-ab0a-a967a2a68e39"]}}, "location": "tab:processed-files", "description": "auto_generated_higlass_view_config"}], "static_headers": [{"lab": {"@id": "/labs/4dn-dcic-lab/", "uuid": "828cd4fe-ebb0-4b36-a94a-d2e3a36cc989", "@type": ["Lab", "Item"], "status": "current", "display_title": "4DN DCIC, HMS", "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin", "role.lab_submitter", "submits_for.828cd4fe-ebb0-4b36-a94a-d2e3a36cc989"]}}, "body": "In Situ Hi-C\n\n
\n In situ Hi-C is a method to detect and quantify the pairwise interactions between chromosome regions across the entire genome. It was developed in 2014 as an improvement over the dilution Hi-C method. Compared to standard dilution Hi-C, this technique reduces the frequency of random ligation because the ligation is performed in situ inside the nucleus, a constrained space, instead of in solution, where DNA fragments are floating freely. In addition, this protocol can be done more quickly in the lab and was the first to introduce the use of 4-cutter restriction enzymes as opposed to the previous 6-cutters, providing higher resolution.\n
\n\nThe protocol involves cross-linking the cells with formaldehyde to form links between physically adjacent DNA regions. The cells are then permeabilized with their nuclei intact. A 4-cutter restriction enzyme is used to digest the chromatin into multiple DNA fragments. The resulting fragments are biotinylated by end filling of the fragments ends. The fragments are then ligated and the DNA is purified and sheared. The biotinylated fragments are pulled down from the solution with streptavidin beads and a library is constructed and sequenced. Analysis of the resulting paired-end short read sequences produces a matrix that shows the number of interactions between different DNA regions.\n
\n\nSee Rao et al., 2014 for more details.\n
\n\n\n In situ Hi-C is a method to detect and quantify the pairwise interactions between chromosome regions across the entire genome. It was developed in 2014 as an improvement over the dilution Hi-C method. Compared to standard dilution Hi-C, this technique reduces the frequency of random ligation because the ligation is performed in situ inside the nucleus, a constrained space, instead of in solution, where DNA fragments are floating freely. In addition, this protocol can be done more quickly in the lab and was the first to introduce the use of 4-cutter restriction enzymes as opposed to the previous 6-cutters, providing higher resolution.\n
\n\nThe protocol involves cross-linking the cells with formaldehyde to form links between physically adjacent DNA regions. The cells are then permeabilized with their nuclei intact. A 4-cutter restriction enzyme is used to digest the chromatin into multiple DNA fragments. The resulting fragments are biotinylated by end filling of the fragments ends. The fragments are then ligated and the DNA is purified and sheared. The biotinylated fragments are pulled down from the solution with streptavidin beads and a library is constructed and sequenced. Analysis of the resulting paired-end short read sequences produces a matrix that shows the number of interactions between different DNA regions.\n
\n\nSee Rao et al., 2014 for more details.\n
\n\n\n In situ Hi-C is a method to detect and quantify the pairwise interactions between chromosome regions across the entire genome. It was developed in 2014 as an improvement over the dilution Hi-C method. Compared to standard dilution Hi-C, this technique reduces the frequency of random ligation because the ligation is performed in situ inside the nucleus, a constrained space, instead of in solution, where DNA fragments are floating freely. In addition, this protocol can be done more quickly in the lab and was the first to introduce the use of 4-cutter restriction enzymes as opposed to the previous 6-cutters, providing higher resolution.\n
\n\nThe protocol involves cross-linking the cells with formaldehyde to form links between physically adjacent DNA regions. The cells are then permeabilized with their nuclei intact. A 4-cutter restriction enzyme is used to digest the chromatin into multiple DNA fragments. The resulting fragments are biotinylated by end filling of the fragments ends. The fragments are then ligated and the DNA is purified and sheared. The biotinylated fragments are pulled down from the solution with streptavidin beads and a library is constructed and sequenced. Analysis of the resulting paired-end short read sequences produces a matrix that shows the number of interactions between different DNA regions.\n
\n\nSee Rao et al., 2014 for more details.\n
\n\n