Optimized ChIP-seq Protocol for H3K27ac
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Corresponding Organization :
Other organizations : Moffitt Cancer Center, Wellcome Sanger Institute, MRC Epidemiology Unit, University of Cambridge, Cedars-Sinai Medical Center, Mayo Clinic in Florida, Duke Medical Center, QIMR Berghofer Medical Research Institute, Université Laval, Centre hospitalier universitaire de Québec, Academic Medical Center, Radboud University Nijmegen, Radboud University Medical Center, Johns Hopkins Medicine, Johns Hopkins University, Chapel Allerton Hospital, Lunenfeld-Tanenbaum Research Institute, University of Toronto, Mount Sinai Hospital, University of California, Irvine, N.N. Alexandrov National Cancer Centre, Hospital Agioi Anargyroi, Kiel University, The University of Texas MD Anderson Cancer Center, Karolinska University Hospital, Karolinska Institutet, Fondazione IRCCS Istituto Nazionale dei Tumori, Vall d'Hebron Hospital Universitari, Royal Marsden Hospital, Centre Léon Bérard, National University Hospital of Iceland, Oregon Health & Science University, Comprehensive Cancer Center Erlangen, Vanderbilt University, Spanish National Cancer Research Centre, Russian Academy of Sciences, Princess Margaret Cancer Centre, University Health Network, Massachusetts General Hospital, Haukeland University Hospital, National Cancer Institute, City Of Hope National Medical Center, City of Hope, Maastricht University, Medizinische Hochschule Hannover, Vejle Sygehus, European Institute of Oncology, Skåne University Hospital, Lund University, University of Pennsylvania, Cancer Research UK Cambridge Center, Royal Devon and Exeter Hospital, Canada's Michael Smith Genome Sciences Centre, BC Cancer Agency, Cancer Council Victoria, Institut d'Investigació Biomèdica de Girona, Institut Català d'Oncologia, Institut Curie, University of Helsinki, Helsinki University Hospital, Huntsman Cancer Institute, Instituto de Investigación Sanitaria del Hospital Clínico San Carlos, Azienda Ospedaliera Universitaria Pisana, Peter MacCallum Cancer Centre, Roswell Park Comprehensive Cancer Center, University of Hawaiʻi at Mānoa, University of California, San Francisco, German Cancer Research Center, Heidelberg University, Westmead Institute for Medical Research, University of Sydney, Columbia University, Ghent University Hospital, University of New Mexico, Sheffield Children's Hospital, Brigham and Women's Hospital, Harvard University, Mayo Clinic, Social and Scientific Systems (United States), Fox Chase Cancer Center, Bashkir State University, The Maria Sklodowska-Curie National Research Institute of Oncology, Memorial Sloan Kettering Cancer Center, Centre d'Investigation Clinique de Nantes, Dartmouth College, University of Pretoria, Centre International de Recherche sur le Cancer, Instituto de Biomedicina y Genética Molecular de Valladolid, Universidad de Valladolid, Friedrich Schiller University Jena, Jena University Hospital, University of Southampton, Royal Marsden NHS Foundation Trust, Institute of Cancer Research, Copenhagen University Hospital, Rigshospitalet, Friedrich-Alexander-Universität Erlangen-Nürnberg, Leipzig University, Manchester Academic Health Science Centre, University of Manchester, Imperial College London, National Centre of Scientific Research "Demokritos", McGill University, Aristotle University of Thessaloniki, Papageorgiou General Hospital, University of Kansas Medical Center, Dana-Farber Cancer Institute, Sheba Medical Center, Centre for Biomedical Network Research on Rare Diseases, University of Würzburg, University College London, Beatson West of Scotland Cancer Centre, University of Utah, National Institutes of Health, Pomeranian Medical University, University of Southern California, University Hospital Cologne, DKFZ-ZMBH Alliance, Centre for Life, Newcastle upon Tyne Hospital, St George's, University of London, University of Copenhagen, Danish Cancer Society, The Netherlands Cancer Institute, Erasmus MC, NorthShore University HealthSystem, University of Chicago, University of British Columbia, Institute of Oncology NN Petrov, Georgetown University Medical Center, Georgetown University, Duke University, Guy's and St Thomas' NHS Foundation Trust, Vilnius University, Aarhus University Hospital, Cancer Prevention Institute of California, University Hospital Carl Gustav Carus, Westmead Hospital, Seoul St. Mary's Hospital, University of Calgary, University of Bergen, Odense University Hospital, Basque Government, Institut d'Investigació Biomédica de Bellvitge, Cancer Center of Hawaii, University of Hawaii System, Asan Medical Center, Ulsan College, Université Grenoble Alpes, Centre Hospitalier Universitaire de Grenoble, Université Paris Sciences et Lettres, Inserm, Texas Southern University, Fraunhofer Project Centre Wolfsburg, Université Claude Bernard Lyon 1, Centre National de la Recherche Scientifique, Centre de Recherche en Cancérologie de Lyon, Vancouver General Hospital, Stanford University, Public Health Ontario, Cancer Research UK Scotland Institute, University of Glasgow, Amsterdam UMC Location VUmc, Klinikum rechts der Isar, Technical University of Munich, University of Melbourne, University of Pittsburgh, Istituto Oncologico Veneto, Istituti di Ricovero e Cura a Carattere Scientifico, St. Michael's Hospital, The University of Texas Health Science Center at Houston, Heinrich Heine University Düsseldorf, Düsseldorf University Hospital, Invitae (United States), National Institute of Oncology, University of Chicago Medical Center, The Ohio State University Comprehensive Cancer Center – Arthur G. James Cancer Hospital and Richard J. Solove Research Institute, Birmingham Women's Hospital, University Medical Center Utrecht, Breast Cancer Now, Piedmont Reference Center for Epidemiology and Cancer Prevention, University of Florence, Aalborg University Hospital, Instituto Português de Oncologia Francisco Gentil, FIRC Institute of Molecular Oncology, Comprehensive Cancer Center Vienna, Medical University of Vienna, NRG Oncology, Western General Hospital, Centre Hospitalier Universitaire de Saint-Étienne, Hôpital Arnaud de Villeneuve, Haifa Medical Center, Dana-Farber Brigham Cancer Center, Centro de Investigación del Cáncer, Oncode Institute, Fred Hutch Cancer Center, National Institute of Environmental Health Sciences, Université Paris-Sud, Glasgow Royal Infirmary, Great Ormond Street Hospital, Institut Paoli-Calmettes, University of South Florida, University Hospital Heidelberg, National Human Genome Research Institute, Subang Jaya Medical Centre, Cancer Research Malaysia, Magee-Womens Hospital, Latvian Biomedical Research and Study Centre, University of Oxford, Hellenic Health Foundation, Beth Israel Deaconess Medical Center, University Medical Center Groningen, University of Groningen, KU Leuven, Charité - Universitätsmedizin Berlin, Fundación Pública Galega de Medicina Xenómica, Servicio Gallego de Salud, Vanderbilt University Medical Center, Churchill Hospital, University Hospital Leipzig, Universität Ulm, Leiden University Medical Center, Women's College Hospital, University of Virginia, UNSW Sydney, Sapienza University of Rome, Seoul National University, Medical University of South Carolina, Yale University
Variable analysis
- Formaldehyde fixation time (10 minutes)
- Sonication time and power (Covaris E220 evolution Focused-Ultrasonicator)
- Antibody used for ChIP (H3K27ac antibody from Diagenode)
- Incubation time and temperature of antibody-bead conjugates with chromatin (overnight at 4°C)
- Yield of 100-300bp genomic DNA fragments
- Quality of ChIP samples assessed by site-specific qPCR prior to next generation sequencing (NGS)
- Cell culture conditions (4 x 15cm dishes of cells)
- Lysis buffer composition (sarkosyl-containing)
- Bead type (blocked magnetic Dynabeads from Life Technologies)
- Washing buffer (RIPA buffer)
- DNA purification method (QIAquick PCR Purification kit)
- Number of independent immunoprecipitations (two)
- Inclusion of input sample (one per cell line)
- Site-specific qPCR to check quality of ChIP samples prior to NGS
- Not explicitly mentioned
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