Die Autoren danken Jan Appel für seine hervorragende technische Unterstützung bei der Schaffung dieses Protokolls. Die Arbeit im Labor Rada-Iglesias stützt sich auf CMMC intramuralen Finanzierung, DFG Research Grants (RA 2547/1-1, RA 2547/2-1 und TE 1007/3-1), Gruppe der UoC fortgeschrittene Forscher Grant und CECAD Grant.

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Genet Dev. 19 (2), 127-134 (2009).</li><li>ENCODE Project Consortium. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+integrated+encyclopedia+of+DNA+elements+in+the+human+genome.">An integrated encyclopedia of DNA elements in the human genome.</a> Nature. 489 (7414), 57-74 (2012).</li><li>Roadmap Epigenomics Consortium. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrative+analysis+of+111+reference+human+epigenomes.">Integrative analysis of 111 reference human epigenomes.</a> Nature. 518 (7539), 317-330 (2015).</li><li>Heintzman, N. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+modifications+at+human+enhancers+reflect+global+cell-type-specific+gene+expression.">Histone modifications at human enhancers reflect global cell-type-specific gene expression.</a> Nature. 459 (7243), 108-112 (2009).</li><li>Rada-Iglesias, A., Bajpai, R., Swigut, T., Brugmann, S. A., Flynn, R. A., Wysocka, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+unique+chromatin+signature+uncovers+early+developmental+enhancers+in+humans.">A unique chromatin signature uncovers early developmental enhancers in humans.</a> Nature. 470 (7333), 279-283 (2011).</li><li>Creyghton, M. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+H3K27ac+separates+active+from+poised+enhancers+and+predicts+developmental+state.">Histone H3K27ac separates active from poised enhancers and predicts developmental state.</a> Proc Nat Acad Sci USA. 107 (50), 21931-21936 (2010).</li><li>Benayoun, B. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=H3K4me3+breadth+is+linked+to+cell+identity+and+transcriptional+consistency.">H3K4me3 breadth is linked to cell identity and transcriptional consistency.</a> Cell. 158 (3), 673-688 (2014).</li><li>Rehimi, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenomics-Based+Identification+of+Major+Cell+Identity+Regulators+within+Heterogeneous+Cell+Populations.">Epigenomics-Based Identification of Major Cell Identity Regulators within Heterogeneous Cell Populations.</a> Cell Rep. 17 (11), 3062-3076 (2016).</li><li>Whyte, W. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Master+transcription+factors+and+mediator+establish+super-enhancers+at+key+cell+identity+genes.">Master transcription factors and mediator establish super-enhancers at key cell identity genes.</a> Cell. 153 (2), 307-319 (2013).</li><li>Valouev, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+analysis+of+transcription+factor+binding+sites+based+on+ChIP-Seq+data.">Genome-wide analysis of transcription factor binding sites based on ChIP-Seq data.</a> Nat Methods. 5 (9), 829-834 (2008).</li><li>Dahl, J. 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C., Bock, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ChIPmentation:+fast,+robust,+low-input+ChIP-seq+for+histones+and+transcription+factors.">ChIPmentation: fast, robust, low-input ChIP-seq for histones and transcription factors.</a> Nat Methods. 12 (10), 963-965 (2015).</li><li>Zhang, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allelic+reprogramming+of+the+histone+modification+H3K4me3+in+early+mammalian+development.">Allelic reprogramming of the histone modification H3K4me3 in early mammalian development.</a> Nature. 537 (7621), 553-557 (2016).</li><li>Hamburger, V., Hamilton, H. 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Die Autoren müssen keine finanzielle Interessenkonflikte offen gelegt werden.

Epigenomischen Profilierung der Histon-Modifikation mit ChIP-Seq kann verwendet werden, zur Verbesserung der funktionellen Annotations vertebrate Genom in verschiedenen zellulären Kontexten4,5,18. Diese epigenomischen-Profile eingesetzt werden, unter anderem um zu identifizieren, Enhancer-Elemente, um die rechtliche Definition des Enhancer (d. h. aktiv, grundiert, oder bereit) und großen Zelle Identität Regulierungsb...

Post-translationalen Modifikationen der Histone sind verschiedene Chromatin-abhängige Prozesse, einschließlich Transkription, Replikation und DNA-Reparatur1,2,3direkt beteiligt. Darüber hinaus verschiedene Histon Änderungen zeigen positive (z. B. H3K4me3 und H3K27ac) oder negativ (z. B. H3K9me3 und H3K27me3) Korrelationen mit Genexpression und kann im großen und ganzen definiert als aktivierende oder repressive Histon markiert, bzw.2,3. Infolgedessen sind globale Histon Änderung Karten, auch bezeichnet als epigenomischen Karten als mächtiges und universelles Werkzeug funktionell vertebrate Genom4,5Anmerkungen zu entstanden. Z. B. distalen regulatorische Sequenzen wie Enhancer identifiziert werden können abhängig vom Vorhandensein von spezifischen Chromatin Unterschriften (z. B. aktive Enhancer: H3K4me1 und H3K27ac), die von proximalen Promoter-Regionen unterscheiden (z.B. aktive Promotoren: H3K4me3)6,7,8. Auf der anderen Seite befinden sich Gene mit großen Zelle Identität regulatorische Funktionen in der Regel mit breiten Chromatin Domains mit H3K4me3 oder H3K27me3, abhängig vom Status der zugrunde liegenden Gene transcriptionally aktiv oder inaktiv gekennzeichnet bzw.9 ,10. Ebenso scheint der Ausdruck der großen Zelle Identität Gene häufig von mehreren und räumlich gesteuert werden gruppierten Enhancer (d. h. Super-Enhancer), die als breite H3K27ac versehenen Domänen11identifiziert werden können.
Derzeit entstehen Histon Änderung Karten mit ChIP-Seq-Technologie, bietet im Vergleich zu bisherigen Ansätzen wie ChIP-Chip (ChIP mit Microarrays gekoppelt) höheren Auflösung, weniger Artefakte, weniger Lärm, mehr Reichweite und geringer kostet12. Dennoch hat die Generation der epigenomischen-Karten mit ChIP-Seq-Technologie seiner inhärenten Einschränkungen, meist verbunden mit der Fähigkeit, erfolgreich ChIP in den Proben von Interesse zu erfüllen. Traditionellen ChIP Protokolle benötigt in der Regel Millionen von Zellen, welche Grenzen der Anwendbarkeit dieser Methode zur in-vitro- Zelle Zeilen oder Zellen, die isoliert in Vivo (z. B. Blutzellen) problemlos. In den letzten Jahren wurden eine Reihe von modifizierten ChIP-Protokollen kompatibel mit niedrigen Zellzahlen beschrieben13,14,15,16. Aber diese Protokolle sind speziell mit Next Generation Sequencing (z.B. ChIP-Seq) gekoppelt werden, und in der Regel verwenden sie ad-hoc- Bibliothek Vorbereitung Methoden13,14, 15 , 16.
Wir beschrieben hier ein ChIP-Protokoll, das verwendet werden kann, zu untersuchen, Histon-Änderung-Profile mit niedrigen bis mittleren Zellzahlen (5 x 104 - 5 x 105 Zellen) an beiden ausgewählten Loci (z.B. ChIP-qPCR) oder Global (d. h. ChIP-Seq) (Abbildung 1). Wenn ChIP-Seq-Technologie gekoppelt, kann unser ChIP-Protokoll zusammen mit Standardbibliothek Zubereitungsmethoden, wodurch es allgemein zugänglich, viele Labors10verwendet werden. Dieses Protokoll wurde verwendet, um mehrere Histon-Marken untersuchen (z. B. H3K4me3, H3K27me3 und H3K27ac) in verschiedenen Huhn embryonalen Geweben (z. B. spinale Neuralrohr (SNT), frontonasalen Protuberanzen und Epiblast). Allerdings erwarten wir, dass es breit anwendbar auf andere Organismen sein sollte, in denen biologisch bzw. klinisch relevante Proben nur in geringen Mengen erreicht werden können.

<table><tbody><tr><td>&lt;strong&gt;Reagent&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>BSA powder</td><td>Carl Roth</td><td>3737.3</td><td></td></tr><tr><td>Phosphate Saline buffer (PBS)</td><td>Sigma Aldrich</td><td>D8537</td><td></td></tr><tr><td>Tris-HCL pH 8.0</td><td>Sigma Aldrich</td><td>T1503</td><td></td></tr><tr><td>NaCl</td><td>Carl Roth</td><td>3957.2</td><td></td></tr><tr><td>EDTA</td><td>Carl Roth</td><td>8043.2</td><td></td></tr><tr><td>EGTA</td><td>Carl Roth</td><td>3054.2</td><td></td></tr><tr><td>Na-Deoxycholate</td><td>Sigma Aldrich</td><td>D6750-24</td><td></td></tr><tr><td>N-lauroylsarcosine</td><td>Sigma Aldrich</td><td>61743-25G</td><td></td></tr><tr><td>Hepes</td><td>Applichem</td><td>A3724,0250</td><td></td></tr><tr><td>LiCl</td><td>Carl Roth</td><td>3739.2</td><td></td></tr><tr><td>NP-40</td><td>Sigma Aldrich</td><td>I3021-100ml</td><td></td></tr><tr><td>SDS</td><td>Carl Roth</td><td>1833</td><td></td></tr><tr><td>Protein G/magnetic beads</td><td>Invitrogen</td><td>1004D</td><td></td></tr><tr><td>37% Formaldehyde</td><td>Sigma Aldrich</td><td>252549-1L</td><td></td></tr><tr><td>Glycine</td><td></td><td></td><td></td></tr><tr><td>RNase</td><td>Peqlab</td><td>12-RA-03</td><td></td></tr><tr><td>Proteinase K</td><td>Sigma Aldrich</td><td>46.35 E</td><td></td></tr><tr><td>Na-butyrate</td><td>Sigma Aldrich</td><td>SLB2659V</td><td></td></tr><tr><td>Proteinase inhibitor</td><td>Roche</td><td>5892791001</td><td></td></tr><tr><td>SYBRgreen Mix</td><td>biozym</td><td>617004</td><td></td></tr><tr><td>dH&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma Aldrich</td><td>W4502</td><td></td></tr><tr><td>1 Kb ladder</td><td>Thermofisher</td><td>SM1333</td><td></td></tr><tr><td>Orange G</td><td>Sigma Alrich</td><td>03756-25</td><td></td></tr><tr><td>Agarose</td><td>Invitrogen</td><td>16500-500</td><td></td></tr><tr><td>0.25% Trypsin-EDTA (1x)</td><td>Gibco</td><td>25200-072</td><td></td></tr><tr><td>Octylphenol Ethoxylate (Triton X 100)</td><td>Roth</td><td>3051.4</td><td></td></tr><tr><td>DMEM (Dulbecco&amp;acute;s Modified Eagle Medium)</td><td>Gibco</td><td>31331-028</td><td></td></tr><tr><td>Gel loading tips Multiflex</td><td>A.Hartenstein</td><td>GS21</td><td></td></tr><tr><td>qPCR Plates</td><td>Sarstedt</td><td>721,985,202</td><td></td></tr><tr><td>384 well</td><td>Sarstedt</td><td>721,985,202</td><td></td></tr><tr><td>1.5 mL tubes</td><td>Sarstedt</td><td>72,706</td><td></td></tr><tr><td>100 - 1,000 &amp;micro;L Filter tips</td><td>Sarstedt</td><td>70,762,211</td><td></td></tr><tr><td>2 - 20 &amp;micro;L Filter tips</td><td>Sarstedt</td><td>70,760,213</td><td></td></tr><tr><td>2 - 200 &amp;micro;L Filter tips</td><td>Sarstedt</td><td>70,760,211</td><td></td></tr><tr><td>0.5 - 10 &amp;micro;L Filter tips</td><td>Sarstedt</td><td>701,116,210</td><td></td></tr><tr><td>H3K27me3 antibody</td><td>Active Motif</td><td>39155</td><td></td></tr><tr><td>H3K27ac antibody</td><td>Active Motif</td><td>39133</td><td></td></tr><tr><td>H3K4me3 antibody</td><td>Active Motif</td><td>39159</td><td></td></tr><tr><td>H3K4me2 antibody</td><td>Active Motif</td><td>39141</td><td></td></tr><tr><td>End Repair Mix</td><td>Illumina</td><td>FC-121-4001</td><td></td></tr><tr><td>Paramagnetic beads</td><td>Beckman Coulter</td><td>A63881</td><td></td></tr><tr><td>Resuspension buffer</td><td>Illumina</td><td>FC-121-4001</td><td></td></tr><tr><td>A-Tailing Mix</td><td>Illumina</td><td>FC-121-4001</td><td></td></tr><tr><td>Ligation Mix</td><td>Illumina</td><td>FC-121-4001</td><td></td></tr><tr><td>RNA Adapter Indexes</td><td>Illumina</td><td>RS-122-2101</td><td></td></tr><tr><td>Stop Ligation Buffer</td><td>Illumina</td><td>FC-121-4001</td><td></td></tr><tr><td>PCR Primer Cocktail</td><td>Illumina</td><td>FC-121-4001</td><td></td></tr><tr><td>Enhanced PCR Mix</td><td>Illumina</td><td>FC-121-4001</td><td></td></tr><tr><td>Genomic DNA ladder</td><td>Agilent</td><td>5067-5582</td><td></td></tr><tr><td>Elution Buffer</td><td>Agilent</td><td>19086</td><td></td></tr><tr><td>Sample Buffer</td><td>Agilent</td><td>5067-5582</td><td></td></tr><tr><td>Library Quantification Kit</td><td>Kapa Biosystems</td><td>KK4835 &amp;ndash; 07960204001</td><td></td></tr><tr><td>Fertile chicken white eggs</td><td>LSL Rhein Main</td><td>KN: 15968</td><td></td></tr><tr><td>Needle (Neoject)</td><td>A.Hartenstein</td><td>10001</td><td></td></tr><tr><td>Syringe (Ecoject 10 mL)</td><td>Dispomed witt oHG</td><td>21010</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Centrifuge</td><td>Hermle</td><td>Z 216 MK</td><td></td></tr><tr><td>Thermoshaker</td><td>ITABIS</td><td>MKR13</td><td></td></tr><tr><td>Sonicator</td><td>Active Motive</td><td>EpiShear probe sonicator</td><td></td></tr><tr><td>Sonicator</td><td>Diagenode</td><td>Bioruptor Plus</td><td></td></tr><tr><td>Rotator</td><td>Stuart</td><td>SB3</td><td></td></tr><tr><td>Variable volume (5 &amp;ndash;1,000 &amp;mu;L) pipettes</td><td>Eppendorf</td><td>N/A</td><td></td></tr><tr><td>Timer</td><td>Sigma</td><td>N/A</td><td></td></tr><tr><td>Magnetic holder</td><td>Thermo Fisher</td><td>DynaMag-2 12321D</td><td></td></tr><tr><td>Table spinner</td><td>Heathrow Scientific</td><td>Sprout</td><td></td></tr><tr><td>Mixer</td><td>LMS</td><td>VTX 3000L</td><td></td></tr><tr><td>Real-Time PCR Cycler</td><td>Roche</td><td>Light Cycler; Serial Nr.5662</td><td></td></tr><tr><td>PCR Cycler</td><td>Applied Biosystems</td><td>Gene Amp PCR System 9700</td><td></td></tr><tr><td>DNA and RNA quality control system</td><td>Agilent</td><td>Agilent 4200 TapeStation System</td><td></td></tr><tr><td>Forceps</td><td>Dumont</td><td>5-Inox-H</td><td></td></tr><tr><td>Perforated spoon</td><td>World precision instruments</td><td>501997</td><td></td></tr></tbody></table>

chromatin immunoprecipitation (chip) protocol for low-abundance embryonic samples

Chromatin Immunopräzipitation (ChIP) ist eine weit verbreitete Technik zur Kartierung der Lokalisierung von posttranslational modifizierte Histone, Histon-Varianten, Transkriptionsfaktoren oder Chromatin-modifizierende Enzyme, die an einen bestimmten Ort oder auf einer genomweiten Skala. Die Kombination von ChIP-Assays mit Next Generation Sequencing (z.B. ChIP-Seq) ist ein leistungsfähiger Ansatz weltweit genregulatorischer Netzwerke aufzudecken und die funktionale Anmerkung von Genomen, insbesondere von nicht-kodierenden regulatorischen Sequenzen zu verbessern. ChIP-Protokolle erfordern normalerweise große Mengen von Zellmaterial, damit die Anwendbarkeit dieser Methode zu untersuchen, seltene Zelltypen oder kleine Gewebebiopsien entgegensteht. Um die ChIP-Assay kompatibel mit der Menge des biologischen Materials vornehmen, die in der Regel in Vivo in der frühen Embryogenese vertebrate gewonnen werden können, beschreiben wir hier eine vereinfachte ChIP-Protokoll, in dem die Anzahl der Schritte erforderlich, um abzuschließen, die Assay wurden reduziert, um die Probenverlust zu minimieren. Dieses ChIP-Protokoll wird erfolgreich eingesetzt, um verschiedene Histon-Modifikationen in verschiedenen embryonalen und adulten Maus Geweben mit niedrigen bis mittleren Zellzahlen (5 x 104 - 5 x 105 Zellen) zu untersuchen. Wichtig ist, dieses Protokolls ist kompatibel mit ChIP-Seq-Technologie mit Standardbibliothek Zubereitungsmethoden, wodurch globale epigenomischen-Karten von hochaktuellen embryonalen Gewebe.

Um die Leistung unserer ChIP-Protokoll zu veranschaulichen, haben wir ChIP-Seq-Experimente mit gepoolten SNT Abschnitte von HH19 Hühnerembryos, maxillaris Protuberanzen HH22 Embryonen, und Bühne-HH3 Hühnerembryos zu untersuchen, die verbindliche Profile von Huhn verschiedenen Histon-Modifikationen (z. B. H3K4me2, H3K27ac, H3K4me3 und H3K27me3). Das gewonnene ChIP DNAs waren wurde die Beschallung Effizienz durch Agarose-Gelelektrophorese der entsprechenden Eingabe DNAs (<strong...

Watch this Scientific Journal Video about Chromatin Immunoprecipitation ChIP Protocol for Low-abundance Embryonic Samples at JoVE.com

Chromatin Immunoprecipitation ChIP Protocol for Low-abundance Embryonic Samples

Hier beschreiben wir eine Chromatin Immunopräzipitation (ChIP) und ChIP-Seq Bibliothek Vorbereitung Protokoll zum globalen epigenomischen Profile aus Low-Fülle Huhn embryonalen Proben zu generieren.

Chromatin Immunopräzipitation (ChIP) Protokoll für Low-Fülle embryonalen Proben

Chromatin immunoprecipitation (ChIP) is a widely-used technique for mapping the localization of post-translationally modified histones, histone variants, ...

chromatin-immunoprecipitation-chip-protocol-for-low-abundance

Universidad de Cartagena UDEC

Research

JoVE Journal

Genetics

Chromatin Immunoprecipitation (ChIP) Protocol for Low-abundance Embryonic Samples

15.6K Aufrufe.  University of Cologne. Hier beschreiben wir eine Chromatin Immunopräzipitation (ChIP) und ChIP-Seq Bibliothek Vorbereitung Protokoll zum globalen epigenomischen Profile aus Low-Fülle Huhn embryonalen Proben zu generieren.

Serie: Chromatin Immunoprecipitation ChIP Protocol for Low-abundance Embryonic Samples

Genome-wide Snapshot of Chromatin Regulators and States in Xenopus Embryos by ChIP-Seq

The recruitment of chromatin regulators and the assignment of chromatin states to specific genomic loci are pivotal to cell fate decisions and tissue and organ formation during development. Determining the locations and levels of such chromatin features in vivo will provide valuable information about the spatio-temporal regulation of genomic elements, and will support aspirations to mimic embryonic tissue development in vitro. The most commonly used method for genome-wide and high-resolution profiling is chromatin immunoprecipitation followed by next-generation sequencing (ChIP-Seq). This protocol outlines how yolk-rich embryos such as those of the frog Xenopus can be processed for ChIP-Seq experiments, and it offers simple command lines for post-sequencing analysis. Because of the high efficiency with which the protocol extracts nuclei from formaldehyde-fixed tissue, the method allows easy upscaling to obtain enough ChIP material for genome-wide profiling. Our protocol has been used successfully to map various DNA-binding proteins such as transcription factors, signaling mediators, components of the transcription machinery, chromatin modifiers and post-translational histone modifications, and for this to be done at various stages of embryogenesis. Lastly, this protocol should be widely applicable to other model and non-model organisms as more and more genome assemblies become available.

Genome-wide Snapshot of Chromatin Regulators and States in Xenopus Embryos by ChIP-Seq

The question of how chromatin regulators and chromatin states affect the genome in vivo is key to our understanding of how early cell fate decisions are made in the developing embryo. ChIP-Seq&#8212;the most popular approach to investigate chromatin features at a global level&#8212;is outlined here for Xenopus embryos.

Genomweite Snapshot von Chromatin Regulierungsbehörden und Staaten in<em&gt; Xenopus</em&gt; Embryonen von ChIP-Seq

The recruitment of chromatin regulators and the assignment of chromatin states to specific genomic loci are pivotal to cell fate decisions and tissue and ...

Forschung

Entwicklungsbiologie

Chromatin Immunoprecipitation (ChIP) in Mouse T-cell Lines

Signaling pathways regulate gene expression programs via the modulation of the chromatin structure at different levels, such as by post-translational modifications (PTMs) of histone tails, the exchange of canonical histones with histone variants, and nucleosome eviction. Such regulation requires the binding of signal-sensitive transcription factors (TFs) that recruit chromatin-modifying enzymes at regulatory elements defined as enhancers. Understanding how signaling cascades regulate enhancer activity requires a comprehensive analysis of the binding of TFs, chromatin modifying enzymes, and the occupancy of specific histone marks and histone variants. Chromatin immunoprecipitation (ChIP) assays utilize highly specific antibodies to immunoprecipitate specific protein/DNA complexes. The subsequent analysis of the purified DNA allows for the identification the region occupied by the protein recognized by the antibody. This work describes a protocol to efficiently perform ChIP of histone proteins in a mature mouse T-cell line. The presented protocol allows for the performance of ChIP assays in a reasonable timeframe and with high reproducibility.

Chromatin Immunoprecipitation ChIP in Mouse T-cell Lines

This work describes a protocol for chromatin immunoprecipitation (ChIP) using a mature mouse T-cell line. This protocol is suitable to investigate the distribution of specific histone marks at specific promoter sites or genome-wide.

Chromatin-Immunpräzipitation (ChIP) in Maus-T-Zell-Linien

Signaling pathways regulate gene expression programs via the modulation of the chromatin structure at different levels, such as by post-translational ...

Genetik

Epigenetic Regulation of Cardiac Differentiation of Embryonic Stem Cells and Tissues

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated by transcription factors which bind genomic regulatory regions including enhancers and promoters of cardiac constitutive genes. DNA is wrapped around histones that are subjected to chemical modifications. Modifications of histones further lead to repressed, activated or poised gene transcription, thus bringing another level of fine tuning regulation of gene transcription. Embryonic Stem cells (ES cells) recapitulate within embryoid bodies (i.e., cell aggregates) or in 2D culture the early steps of cardiac development. They provide in principle enough material for chromatin immunoprecipitation (ChIP), a technology broadly used to identify gene regulatory regions. Furthermore, human ES cells represent a human cell model of cardiogenesis. At later stages of development, mouse embryonic tissues allow for investigating specific epigenetic landscapes required for determination of cell identity. Herein, we describe protocols of ChIP, sequential ChIP followed by PCR or ChIP-sequencing using ES cells, embryoid bodies and cardiac specific embryonic regions. These protocols allow to investigating the epigenetic regulation of cardiac gene transcription.

A fine tuning regulation of gene transcription underlies embryonic cell fate decision. Herein, we describe chromatin immunoprecipitation assays used to investigate epigenetic regulation of both cardiac differentiation of stem cells and cardiac development of mouse embryos.

Epigenetische Regulation der Herz Differenzierung von embryonalen Stammzellen und Gewebe

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated ...

Chromatin Immunoprecipitation of Murine Brown Adipose Tissue

Most cellular processes are regulated by transcriptional modulation of specific gene programs. Such modulation is achieved through the combined actions of a wide range of transcription factors (TFs) and cofactors mediating transcriptional activation or repression via changes in chromatin structure. Chromatin immunoprecipitation (ChIP) is a useful molecular biology approach for mapping histone modifications and profiling transcription factors/cofactors binding to DNA, thus providing a snapshot of the dynamic nuclear changes occurring during different biological processes.
To study transcriptional regulation in adipose tissue, samples derived from in vitro cell cultures of immortalized or primary cell lines are often favored in ChIP assays because of the abundance of starting material and reduced biological variability. However, these models represent a limited snapshot of the actual chromatin state in living organisms. Thus, there is a critical need for optimized protocols to perform ChIP on adipose tissue samples derived from animal models.
Here we describe a protocol for efficient ChIP-seq of both histone modifications and non-histone proteins in brown adipose tissue (BAT) isolated from a mouse. The protocol is optimized for investigating genome-wide localization of proteins of interest and epigenetic markers in the BAT, which is a morphologically and physiologically distinct tissue amongst fat depots.

Here we describe a protocol for efficient chromatin immunoprecipitation (ChIP) followed by high-throughput DNA sequencing (ChIP-seq) of brown adipose tissue (BAT) isolated from a mouse. This protocol is suitable for both mapping histone modifications and investigating genome-wide localization of non-histone proteins of interest in vivo.

Chromatin Immunopräzipitation der murinen braunen Fettgewebe

Most cellular processes are regulated by transcriptional modulation of specific gene programs. Such modulation is achieved through the combined actions of ...

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

Chromatin Immunopräzipitation von humanen embryonalen Stammzellen

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

Biologie

CARIP-Seq and ChIP-Seq: Methods to Identify Chromatin-Associated RNAs and Protein-DNA Interactions in Embryonic Stem Cells

Embryonic stem (ES) cell self-renewal&#160;and differentiation is governed by extrinsic signals and intrinsic networks of transcription factors, epigenetic regulators, and post-translation modifications of histones that combinatorially influence the gene expression state of nearby genes. RNA has also been shown to interact with various proteins to regulate chromatin dynamics&#160;and gene expression. Chromatin-associated RNA immunoprecipitation (CARIP) followed by next-generation sequencing (CARIP-Seq) is a novel method to survey RNAs associated with chromatin proteins, while chromatin immunoprecipitation followed by next-generation sequencing (ChIP-Seq) is a powerful genomics technique to map the location of post-translational modification of histones, transcription factors, and epigenetic modifiers on a global-scale in ES cells. Here, we describe methods to perform CARIP-Seq and ChIP-Seq, including library construction for next-generation sequencing, to generate global chromatin-associated RNA and epigenomic maps in ES cells.

Here, we describe methods to perform ChIP-Seq and CARIP-Seq, including library preparation for next-generation sequencing, to generate global epigenomic and chromatin-associated RNA maps in ES cells.

CARIP-Seq und ChIP-Seq: Methoden zur Ermittlung von Chromatin-assoziierter RNA und Protein-DNA-Wechselwirkungen in embryonale Stammzellen

Embryonic stem (ES) cell self-renewal&#160;and differentiation is governed by extrinsic signals and intrinsic networks of transcription factors, ...

Chromatin Immunoprecipitation Assay for Tissue-specific Genes using Early-stage Mouse Embryos

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This technique has been widely exploited in tissue culture cells, and to a lesser extent, in primary tissue. The application of ChIP to rodent embryonic tissue, especially at early times of development, is complicated by the limited amount of tissue and the heterogeneity of cell and tissue types in the embryo. Here we present a method to perform ChIP using a dissociated embryonic day 8.5 (E8.5) embryo. Sheared chromatin from a single E8.5 embryo can be divided into up to five aliquots, which allows the investigator sufficient material for controls and for investigation of specific protein:chromatin interactions.
We have utilized this technique to begin to document protein:chromatin interactions during the specification of tissue-specific gene expression programs. The heterogeneity of cell types in an embryo necessarily restricts the application of this technique because the result is the detection of protein:chromatin interactions without distinguishing whether the interactions occur in all, a subset of, or a single cell type(s). However, examination of tissue-specific genes during or following the onset of tissue-specific gene expression is feasible for two reasons. First, immunoprecipitation of tissue specific factors necessarily isolates chromatin from the cell type where the factor is expressed. Second, immunoprecipitation of coactivators and histones containing post-translational modifications that are associated with gene activation should only be found at genes and gene regulatory sequences in the cell type where the gene is being or has been activated. The technique should be applicable to the study of most tissue-specific gene activation events.
In the example described below, we utilized E8.5 and E9.5 mouse embryos to examine factor binding at a skeletal muscle specific gene promoter. Somites, which are the precursor tissues from which the skeletal muscles of the trunk and limbs will form, are present at E8.5-9.54,5. Myogenin is a regulatory factor required for skeletal muscle differentiation 6-9. The data demonstrate that myogenin is associated with its own promoter in E8.5 and E9.5 embryos. Because myogenin is only expressed in somites at this stage of development 6,10, the data indicate that myogenin interactions with its own promoter have already occurred in skeletal muscle precursor cells in E8.5 embryos.

We demonstrate a chromatin immunoprecipitation (ChIP) method to identify factor interactions at tissue-specific genes during or after the onset of tissue-specific gene expression in mouse embryonic tissue. This protocol should be widely applicable for the study of tissue-specific gene activation as it occurs during normal embryonic development.

Chromatin Immunopräzipitation-Assay für Tissue-spezifische Gene mit Frühzeitige muriner Embryonen

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This ...

Isolation and Cultivation of Neural Progenitors Followed by Chromatin-Immunoprecipitation of Histone 3 Lysine 79 Dimethylation Mark

Brain development is a complex process, which is controlled in a temporo-spatial manner by gradients of morphogens and different transcriptional programs. Additionally, epigenetic chromatin modifications, like histone methylation, have an important role for establishing and maintaining specific cell fates within this process. The vast majority of histone methylation occurs on the flexible histone tail, which is accessible to histone modifiers, erasers, and histone reader proteins. In contrast, H3K79 methylation is located in the globular domain of histone 3 and is implicated in different developmental functions. H3K79 methylation is evolutionarily conserved and can be found in a wide range of species from Homo sapiens to Saccharomyces cerevisiae. The modification occurs in different cell populations within organisms, including neural progenitors. The location of H3K79 methylation in the globular domain of histone 3 makes it difficult to assess. Here, we present methods to isolate and culture cortical progenitor cells (CPCs) from embryonic cortical brain tissue (E11.5-E14.5) or cerebellar granular neuron progenitors (CGNPs) from postnatal tissue (P5-P7), and to efficiently immunoprecipitate H3K79me2 for quantitative PCR (qPCR) and genome-wide sequencing.

We present an effective and reproducible method to isolate and culture neural progenitor cells from embryonic and postnatal brain tissue for chromatin immunoprecipitation (ChIP) of histone 3 lysine 79 dimethylation (H3K79me2) - a histone mark located within the globular domain of histone 3.

Isolierung und Kultivierung von neuronalen Vorläuferzellen gefolgt von Chromatin Immunopräzipitation Histon 3 Lysin 79 Dimethylation Marke

Brain development is a complex process, which is controlled in a temporo-spatial manner by gradients of morphogens and different transcriptional programs. ...

Neurowissenschaften

Efficient Chromatin Immunoprecipitation using Limiting Amounts of Biomass

Chromatin immunoprecipitation (ChIP) is a widely-used method for determining the interactions of different proteins with DNA in chromatin of living cells. Examples include sequence-specific DNA binding transcription factors, histones and their different modification states, enzymes such as RNA polymerases and ancillary factors, and DNA repair components. Despite its ubiquity, there is a lack of up-to-date, detailed methodologies for both bench preparation of material and for accurate analysis allowing quantitative metrics of interaction. Due to this lack of information, and also because, like any immunoprecipitation, conditions must be re-optimized for new sets of experimental conditions, the ChIP assay is susceptible to inaccurate or poorly quantitative results.
Our protocol is ultimately derived from seminal work on transcription factor:DNA interactions1,2 , but incorporates a number of improvements to sensitivity and reproducibility for difficult-to-obtain cell types. The protocol has been used successfully3,4 , both using qPCR to quantify DNA enrichment, or using a semi-quantitative variant of the below protocol.
This quantitative analysis of PCR-amplified material is performed computationally, and represents a limiting factor in the assay. Important controls and other considerations include the use of an isotype-matched antibody, as well as evaluation of a control region of genomic DNA, such as an intergenic region predicted not to be bound by the protein under study (or anticipated not to show changes under the experimental conditions). In addition, a standard curve of input material for every ChIP sample is used to derive absolute levels of enrichment in the experimental material. Use of standard curves helps to take into account differences between primer sets, regardless of how carefully they are designed, and also efficiency differences throughout the range of template concentrations for a single primer set. Our protocol is different from others that are available5-8 in that we extensively cover the later, analysis phase.

We describe a robust method for chromatin immunoprecipitation using primary T cells. The method is founded on standard approaches, but uses a specific set of conditions and reagents that improve efficiency for limited a quantities of cells. Importantly, a detailed description of the data analysis phase is presented.

Effiziente Chromatin Immunopräzipitation mit limitierende Mengen an Biomasse

Chromatin immunoprecipitation (ChIP) is a  widely-used method for determining the interactions of different proteins with  DNA in chromatin of living ...

TChIP-Seq: Cell-Type-Specific Epigenome Profiling

Epigenetic regulation plays central roles in gene expression. Since histone modification was discovered in the 1960s, its physiological and pathological functions have been extensively studied. Indeed, the advent of next-generation deep sequencing and chromatin immunoprecipitation (ChIP) via specific histone modification antibodies has revolutionized our view of epigenetic regulation across the genome. Conversely, tissues typically consist of diverse cell types, and their complex mixture poses analytic challenges to investigating the epigenome in a particular cell type. To address the cell type-specific chromatin state in a genome-wide manner, we recently developed tandem chromatin immunoprecipitation sequencing (tChIP-Seq), which is based on the selective purification of chromatin by tagged core histone proteins from cell types of interest, followed by ChIP-Seq. The goal of this protocol is the introduction of best practices of tChIP-Seq. This technique provides a versatile tool for tissue-specific epigenome investigation in diverse histone modifications and model organisms.

We describe a step-by-step protocol for tandem chromatin immunoprecipitation sequencing (tChIP-Seq) that enables the analysis of cell-type-specific genome-wide histone modification.

TChIP-Seq: Cell-Type-Specific Epigenom Profilierung

Epigenetic regulation plays central roles in gene expression. Since histone modification was discovered in the 1960s, its physiological and pathological ...