{"lab": {"status": "current", "correspondence": [{"contact_email": "YmlyZW5AdWNzZC5lZHU=", "@id": "/users/e3159ffc-a5a9-43a1-8cfa-90b776c39788/", "display_title": "Bing Ren"}], "@id": "/labs/bing-ren-lab/", "@type": ["Lab", "Item"], "title": "Bing Ren, UCSD", "display_title": "Bing Ren, UCSD", "uuid": "795847de-20b6-4f8c-ba8d-185215469cbf", "pi": {"error": "no view permissions"}, "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin", "role.lab_submitter", "submits_for.795847de-20b6-4f8c-ba8d-185215469cbf"]}}, "award": {"@type": ["Award", "Item"], "center_title": "5UM1HL128773-04", "project": "CvDC", "description": "A majority of genetic variants associated with congenital heart disease (CHD) are located within noncoding regions likely to be gene regulatory regions. Therefore, functional validation of enhancers driving expression of CHD relevant genes will lay the groundwork for understanding potentially deleterious variants (PDVs). Enhancers drive tissue specific expression, and cardiovascular (CV) enhancers are poorly conserved between humans and other species. Therefore, it is important to define enhancer function in defined human CV lineages. The hypothesis of our Main Project is that enhancers operate dynamically in a temporal and/or lineage specific manner during cardiomyocyte (CM) specification, and that mutation of enhancers driving expression of CHD relevant genes will disrupt CM lineage development. Aims are: 1) To investigate the transcriptional profile and genome-wide epigenetic landscape of human ventricular and atrial CM lineages during development; 2) To investigate enhancer-promoter interactions that regulate ventricular and atrial gene programs; 3) To examine effects of enhancer mutation on expression of cognate CHD relevant transcription factors and CM lineage development in vitro and in vivo. The Main Project will provide a CV epigenomic regulatory framework for interrogation of PDVs in noncoding regions, as proposed in the Collaborative Project. The hypothesis of our Collaborative Project is that many genetically unexplained CHDs will be due to PDVs which are located in non-coding sequences and perturb target gene expression by altering functions of cis-regulatory elements, thus leading to an overall disruption of pathways critical for normal cardiac lineage development. Aims are: 1) To identify a comprehensive set of candidate CHD non-coding PDVs through systematic analysis of Pediatric Cardiac Genetics Center (PCGC) CHD Whole Genomic Sequencing studies with Cardiovascular Development Center (CvDC) lineage specific human epigenomic datasets; 2) To investigate whether candidate CHD non-coding variants perturb enhancer activity using multiple highthroughput in vitro assays; 3) To investigate the functional significance of high confidence regulatory CHD variants in vitro and in vivo. All aims will utilize hESC models of CV lineage differentiation, and Aim 3 of each project will additionally utilize mouse models. We will establish pipelines of genomewide and highthroughput functional assays, many available through a UCSD Genomics Core. Results will be compared to patient data to investigate congruence of biochemical and morphological/physiological phenotypes. These studies represent collaboration between multiple PIs of complementary expertise, including members of the CvDC and the PCGC (see attached letters), representing an ideal training environment for junior scientists in collaborativ science. Results will result in comprehensive definition of human enhancers dynamically active during differentiation of distinct CV lineages, and will identify variants within these enhancers which contribute to CHD, on a scale made possible only recently by state-of-the-art genomics technologies.", "display_title": "DEVELOPMENTAL REGULATORY GENOMICS OF HUMAN CARDIOVASCULAR LINEAGES", "status": "current", "uuid": "d61f0a46-7e58-477f-b23d-198854b11276", "@id": "/awards/5UM1HL128773-04/", "name": "5UM1HL128773-04", "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin"]}}, "study": "TBX5 and cardiac differentiation", "status": "released", "aliases": ["bruneau-lab:wtc-11-control-day11"], "accession": "4DNESREEJFEH", "condition": "Crispr treated, unmodified control WTC-11-derived cardiac myocytes - differentiation day 11", "description": "Replicates of in situ Hi-C on control Crispr treated but unmodified WTC-11 iPSC cells differentiated for 11 days to cardiac myocytes", "study_group": "Disrupted or Atypical Cells", "viewable_by": ["4DN"], "date_created": "2019-11-27T19:05:41.091881+00:00", "submitted_by": {"error": "no view permissions"}, "dataset_group": "Ren lab TBX5allelic series", "dataset_label": "in situ Hi-C on an allelic series of iPSCs differentiated to cardiac cells", "last_modified": {"modified_by": {"error": "no view permissions"}, "date_modified": "2025-04-02T17:46:31.718157+00:00"}, "public_release": "2025-01-16", "replicate_exps": [{"bio_rep_no": 1, "tec_rep_no": 1, "replicate_exp": {"@type": ["ExperimentHiC", "Experiment", "Item"], "uuid": "b81737b3-ec89-4cbf-b44f-09848487c492", "@id": "/experiments-hi-c/4DNEX22BTA85/", "accession": "4DNEX22BTA85", "display_title": "in situ Hi-C on WTC-11 differentiated to primitive cardiac myocyte with MboI - 4DNEX22BTA85", "status": "released", "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin"]}}}, {"bio_rep_no": 2, "tec_rep_no": 1, "replicate_exp": {"@type": ["ExperimentHiC", "Experiment", "Item"], "uuid": "9b7e6a3c-cce1-4017-b8e2-466d22860a24", "@id": "/experiments-hi-c/4DNEXZJIHVQW/", "accession": "4DNEXZJIHVQW", "display_title": "in situ Hi-C on WTC-11 differentiated to primitive cardiac myocyte with MboI - 4DNEXZJIHVQW", "status": "released", "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin"]}}}], "schema_version": "2", "static_content": [{"content": {"award": {"uuid": "d61f0a46-7e58-477f-b23d-198854b11276", "@id": "/awards/5UM1HL128773-04/", "status": "current", "@type": ["Award", "Item"], "display_title": "DEVELOPMENTAL REGULATORY GENOMICS OF HUMAN CARDIOVASCULAR LINEAGES", "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin"]}}, "uuid": "4a1287db-9574-4420-9718-a867555ca6e7", "title": "4DNESREEJFEH - Processed files", "contributing_labs": [{"uuid": "4dfac8f5-a868-4b29-9c11-a0932130e121", "status": "current", "@type": ["Lab", "Item"], "display_title": "Benoit Bruneau, UCSF", "@id": "/labs/benoit-bruneau-lab/", "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin", "role.lab_submitter", "submits_for.4dfac8f5-a868-4b29-9c11-a0932130e121"]}}], "name": "4a1287db-9574-4420-9718-a867555ca6e7", "@id": "/higlass-view-configs/4a1287db-9574-4420-9718-a867555ca6e7/", "@type": ["HiglassViewConfig", "UserContent", "Item"], "status": "released", "display_title": "4DNESREEJFEH - Processed files", "description": "4DNESREEJFEH (Replicates of in situ Hi-C on control Crispr treated but unmodified WTC-11 iPSC cells differentiated for 11 days to cardiac myocytes): 4DNFIYN1QEJU, 4DNFIRNJKB89, 4DNFIHU4JFWF, 4DNFI3VY4MHM", "filetype": "HiglassViewConfig", "lab": {"status": "current", "uuid": "795847de-20b6-4f8c-ba8d-185215469cbf", "display_title": "Bing Ren, UCSD", "@type": ["Lab", "Item"], "@id": "/labs/bing-ren-lab/", "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin", "role.lab_submitter", "submits_for.795847de-20b6-4f8c-ba8d-185215469cbf"]}}, "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": {"uuid": "828cd4fe-ebb0-4b36-a94a-d2e3a36cc989", "display_title": "4DN DCIC, HMS", "@type": ["Lab", "Item"], "@id": "/labs/4dn-dcic-lab/", "status": "current", "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin", "role.lab_submitter", "submits_for.828cd4fe-ebb0-4b36-a94a-d2e3a36cc989"]}}, "body": "<b>In Situ Hi-C</b>\n\n<p>\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 <i>in situ</i> 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</p>\n<p>\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. 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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 <i>in situ</i> 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</p>\n<p>\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</p>\n<p>\nSee <a href=\"https://www.sciencedirect.com/science/article/pii/S0092867414014974?via%3Dihub\">Rao et al., 2014</a> for more details.\n</p>\n\n<div>\n<img style=\"width: 600px;max-width:100%;\" src=\"https://s3.amazonaws.com/4dn-dcic-public/static-pages/InfoBoxes/IsHC_fig1.png\"/>\n  <br/><br/>\n  <em>Image source: Rao et al., 2014, Figure 1A</em>\n</div>", "filetype": "html", "content_as_html": "<div class=\"html-container\"><b>In Situ Hi-C</b>\n<p>\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 <i>in situ</i> 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</p>\n<p>\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. 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This duplication does not affect the higlass display of these files, howevver, downstream analyses using this file may encounter issues due to this pixel duplication.  The counts from the duplicate pixels can be aggregated to determine the correct count value at that location. If this issue is problematic for your needs you should consider regenerating the matrices from the merged pairs file of the associated dataset using a more recent version of cooler.  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