Wir danken dem Mitglied des Rhee-Labors für den Austausch unveröffentlichter Daten und wertvoller Diskussionen. Diese Arbeit wurde vom Natural Sciences and Engineering Research Council of Canada (NSERC) Stipendium RGPIN-2018-06404 (H.R.) unterstützt.

<ol>
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C., 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+Organization+of+GATA1+and+TAL1+Determined+at+High+Resolution.">Genome-Wide Organization of GATA1 and TAL1 Determined at High Resolution.</a> Molecular and Cellular Biology. 36 (1), 157-172 (2016).</li><li>Rhee, H. S., Bataille, A. R., Zhang, L. Y., Pugh, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subnucleosomal+Structures+and+Nucleosome+Asymmetry+across+a+Genome.">Subnucleosomal Structures and Nucleosome Asymmetry across a Genome.</a> Cell. 159 (6), 1377-1388 (2014).</li><li>Rhee, H. S., Pugh, B. F. <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+structure+and+organization+of+eukaryotic+pre-initiation+complexes.">Genome-wide structure and organization of eukaryotic pre-initiation complexes.</a> Nature. 483 (7389), 295-301 (2012).</li><li>Zhou, X. 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S., 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=Expression+of+Terminal+Effector+Genes+in+Mammalian+Neurons+Is+Maintained+by+a+Dynamic+Relay+of+Transient+Enhancers.">Expression of Terminal Effector Genes in Mammalian Neurons Is Maintained by a Dynamic Relay of Transient Enhancers.</a> Neuron. 92 (6), 1252-1265 (2016).</li><li>Pugh, B. F., Venters, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+Organization+of+Human+Transcription+Initiation+Complexes.">Genomic Organization of Human Transcription Initiation Complexes.</a> Plos One. 11 (2), (2016).</li><li>Wang, S., 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=ATF4+Gene+Network+Mediates+Cellular+Response+to+the+Anticancer+PAD+Inhibitor+YW3-56+in+Triple-Negative+Breast+Cancer+Cells.">ATF4 Gene Network Mediates Cellular Response to the Anticancer PAD Inhibitor YW3-56 in Triple-Negative Breast Cancer Cells.</a> Molecular Cancer Therapeutics. 14 (4), 877-888 (2015).</li><li>Barfeld, S. J., 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=c-Myc+Antagonises+the+Transcriptional+Activity+of+the+Androgen+Receptor+in+Prostate+Cancer+Affecting+Key+Gene+Networks.">c-Myc Antagonises the Transcriptional Activity of the Androgen Receptor in Prostate Cancer Affecting Key Gene Networks.</a> Ebiomedicine. 18, 83-93 (2017).</li><li>McHaourab, Z. F., Perreault, A. A., Venters, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ChIP-seq+and+ChIP-exo+profiling+of+Pol+II,+H2A.Z,+and+H3K4me3+in+human+K562+cells.">ChIP-seq and ChIP-exo profiling of Pol II, H2A.Z, and H3K4me3 in human K562 cells.</a> Scientific Data. 5, 180030 (2018).</li><li>Perreault, A. A., Sprunger, D. M., Venters, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+and+transcriptional+profiling+of+triple+negative+breast+cancer.">Epigenetic and transcriptional profiling of triple negative breast cancer.</a> Scientific Data. 6, 190033 (2019).</li><li>Rossi, M. J., Lai, W. K. M., Pugh, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simplified+ChIP-exo+assays.">Simplified ChIP-exo assays.</a> Nature Communications. 9 (1), 2842 (2018).</li><li>Perreault, A. A., Venters, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ChIP-exo+Method:+Identifying+Protein-DNA+Interactions+with+Near+Base+Pair+Precision.">The ChIP-exo Method: Identifying Protein-DNA Interactions with Near Base Pair Precision.</a> Journal of Visualized Experiments. (118), (2016).</li><li>Jezek, M., Jacques, A., Jaiswal, D., Green, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+Immunoprecipitation+(ChIP)+of+Histone+Modifications+from+Saccharomyces+cerevisiae.">Chromatin Immunoprecipitation (ChIP) of Histone Modifications from Saccharomyces cerevisiae.</a> Journal of Visualized Experiments. (130), (2017).</li><li>Terranova, C., 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=An+Integrated+Platform+for+Genome-wide+Mapping+of+Chromatin+States+Using+High-throughput+ChIP-sequencing+in+Tumor+Tissues.">An Integrated Platform for Genome-wide Mapping of Chromatin States Using High-throughput ChIP-sequencing in Tumor Tissues.</a> Journal of Visualized Experiments. (134), (2018).</li><li>Pchelintsev, N. A., Adams, P. D., Nelson, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+Parameters+for+Efficient+Sonication+and+Improved+Chromatin+Immunoprecipitation+of+High+Molecular+Weight+Proteins.">Critical Parameters for Efficient Sonication and Improved Chromatin Immunoprecipitation of High Molecular Weight Proteins.</a> PLos One. 11 (1), (2016).</li><li>Yamada, N., Lai, W. K. M., Farrell, N., Pugh, B. F., Mahony, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterizing+protein-DNA+binding+event+subtypes+in+ChIP-exo+data.">Characterizing protein-DNA binding event subtypes in ChIP-exo data.</a> Bioinformatics. 35 (6), 903-913 (2019).</li><li>Yen, K., Vinayachandran, V., Pugh, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SWR-C+and+INO80+chromatin+remodelers+recognize+nucleosome-free+regions+near++1+nucleosomes.">SWR-C and INO80 chromatin remodelers recognize nucleosome-free regions near +1 nucleosomes.</a> Cell. 154 (6), 1246-1256 (2013).</li><li>Vinayachandran, V., 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=Widespread+and+precise+reprogramming+of+yeast+protein-genome+interactions+in+response+to+heat+shock.">Widespread and precise reprogramming of yeast protein-genome interactions in response to heat shock.</a> Genome Research. , (2018).</li></ol>

Die Autoren haben nichts zu verraten.

In diesem Protokoll wird ChIP, gefolgt von Exonuklease-Verdauung, verwendet, um DNA-Bibliotheken zur Identifizierung von Protein-DNA-Wechselwirkungen in Säugetierzellen mit ultrahoher Kartierungsauflösung zu erhalten. Viele Variablen tragen zur Qualität des ChIP-exo-Experiments bei. Zu den kritischen experimentellen Parametern gehören die Qualität von Antikörpern, die Optimierung der Beschallung und die Anzahl der LM-PCR-Zyklen. Diese kritischen experimentellen Parameter sind auch, was ChIP-exo-Experimente begrenze...

Die Standorte von Protein-DNA-Wechselwirkungen geben Einblick in die Mechanismen der Genregulation. Chromatin-Immunpräzipitation in Verbindung mit Hochdurchsatz-Sequenzierung (ChIP-seq) wird seit einem Jahrzehnt verwendet, um genomweite Protein-DNA-Wechselwirkungen in lebenden Zellen1,2zu untersuchen. Die ChIP-seq-Methode wird jedoch durch Heterogenität bei der DNA-Fragmentierung und ungebundene DNA-Kontamination eingeschränkt, die zu einer niedrigen Mapping-Auflösung, falschen Positivmeldungen, verpassten Anrufen und einem unspezifischen Hintergrundsignal führen. Die Kombination von ChIP mit Exonuklease-Verdauung (ChIP-exo) verbessert die ChIP-seq-Methode, indem ChIP-DNA auf die Protein-DNA-Vernetzungspunkte gestutzt wird, was eine nahezu Base-Pair-Auflösung und ein niedriges Hintergrundsignal3,4,5bietet. Die stark verbesserte Mapping-Auflösung und der geringe Hintergrund von ChIP-exo ermöglichen die Deko-Lösung präziser und umfassender Protein-DNA-Bindungsstellen im gesamten Genom. ChIP-exo ist in der Lage, funktionell unterschiedliche DNA-bindende Motive, kooperative Wechselwirkungen zwischen Transkriptionsfaktoren (TF) und mehrere Protein-DNA-Vernetzungsstellen an einem bestimmten genomischen Bindungsort zu offenbaren, die durch andere genomische Kartierungsmethodennichtnachweisbar sind 3,4,6,7.
ChIP-exo wurde ursprünglich in angehender Hefe verwendet, um die sequenzspezifische DNA-Bindung von TFs zu untersuchen, um die genaue Organisation des Transkriptions-Vorinitiierungskomplexes und die subnukleosomale Struktur einzelner Histonen im Genom4,8,9zu untersuchen. Seit seiner Einführung im Jahr 20114, ChIP-exo wurde erfolgreich in vielen anderen Organismen einschließlich Bakterien, Mäuse, und menschliche Zellen7,10,11,12,13,14,15,16,17. Im Jahr 2016 verwendeten Rhee et al.14 erstmals ChIP-exo in Säugetierneuronen, um zu verstehen, wie die neuronale Genexpression nach der Downregulation der Programmierung TF Lhx3 aufrechterhalten wurde, die mit einer anderen Programmier-TF Isl1 einen Heterodimer-Komplex bildet. Diese Studie zeigte, dass in Abwesenheit von Lhx3, Isl1 zu neuen neuronalen Enhancer rekrutiert wird, die von Onecut1 TF gebunden sind, um die Genexpression neuronaler Effektorgene aufrechtzuerhalten. In dieser Studie zeigte ChIP-exo, wie mehrere TFs Zelltyp- und zellstufenspezifische DNA-Regulierungselemente in kombinatorischem Weise bei nahezu nukleotid-Mapping-Auflösung dynamisch erkennen. Andere Studien verwendeten auch die ChIP-exo-Methode, um das Zusammenspiel von Proteinen und DNA in anderen Säugetierzelllinien zu verstehen. Han et al.7 verwendeten ChIP-exo, um die genomweite Organisation von GATA1 und TAL1 TFs in Mauserythroidzellen mit ChIP-exo zu untersuchen. Diese Studie ergab, dass TAL1 direkt für die DNA rekrutiert wird und nicht indirekt durch Protein-Protein-Wechselwirkungen mit GATA1 während der Erythroid-Differenzierung. Neuere Studien verwendeten auch ChIP-exo, um die genomweiten Bindungsstellen von CTCF, RNA Polymerase II und Histonmarkierungen zu profilieren, um epigenomische und transkriptionelle Mechanismen in menschlichen Zelllinien18,19zu untersuchen.
Es gibt mehrere Versionen des ChIP-exo Protokolls verfügbar3,5,20. Diese ChIP-exo-Protokolle sind jedoch für Forscher, die mit der Vorbereitung der Sequenzierungsbibliothek der nächsten Generation nicht vertraut sind, schwer zu befolgen. Eine ausgezeichnete Version des ChIP-exo-Protokolls wurde mit leicht verständlichen Anweisungen und einem Video21veröffentlicht, enthielt aber viele enzymatische Schritte, die eine erhebliche Zeitinanspruch samten. Hier berichten wir über eine neue Version des ChIP-exo-Protokolls mit reduzierten enzymatischen Schritten und Inkubationszeiten sowie Erläuterungen für jeden enzymatischen Schritt21. Endreparatur- und dA-Tailing-Reaktionen werden in einem einzigen Schritt mit Endvorbereitungsenzym kombiniert. Die Inkubationszeiten für Index- und Universaladapterligationsschritte werden mit einem Ligationsverstärker von 2 h auf 15 min reduziert. Die Kinase-Reaktion nach dem im vorherigen ChIP-exo-Protokoll beschriebenen Indexadapter-Ligationsschritt wird entfernt. Stattdessen wird während der Oligo-DNA-Synthese eine Phosphatgruppe zu einem der 5' Enden des Indexadapters (Tabelle 1) hinzugefügt, der für den Lambda-Exonuklease-Verdauungsschritt verwendet wird. Während das vorherige ChIP-exo-Protokoll RecJf Exonuclease-Verdauung verwendet, um einsträngige DNA-Verunreinigungen zu beseitigen, wird dieser Verdauungsschritt hier entfernt, da er für die Qualität der ChIP-exo-Bibliothek nicht entscheidend ist. Zur Reinigung der reverse-vernetzten DNA nach der ChIP-Elution wird anstelle der Extraktionsmethode phenol:chloroform:isoamyl alcohol (PCIA) eine Methode zur Reinigung magnetischer Perlen verwendet. Dies reduziert die Inkubationszeit der DNA-Extraktion. Wichtig ist, dass die Mehrheit der Adapterdimer entfernt wird, die während der Indexadapterligation gebildet werden, was die Effizienz der ligationsvermittelten PCR beeinträchtigen kann.
Das hier vorgestellte ChIP-exo-Protokoll ist für den Nachweis präziser Protein-DNA-Wechselwirkungen in Säugetierneuronen optimiert, die sich von Maus-Embryonalstammzellen (ES) unterscheiden. Kurz werden geerntete und vernetzte neuronale Zellen lysiert, damit Chromatin der Beschallung ausgesetzt und dann beschallt werden kann, so dass dna-Fragmente in angemessener Größe erhalten werden (Abbildung 1). Antikörperbeschichtete Perlen werden dann verwendet, um fragmentiertes, lösliches Chromatin selektiv zu immunprezipitieren, um das Protein von Interesse zu erhalten. Während sich die immunpräzipierte DNA noch auf den Perlen befindet, werden Endreparatur, Ligation von Sequenzierungsadaptern, Einfüllreaktion und 5' bis 3' Lambda-Exonuklease-Verdauungsschritte durchgeführt. Der Exonuklease-Verdauungsschritt verleiht ChIP-exo sein ultrahohe Auflösungs- und ein hohes Signal-Rausch-Verhältnis. Lambda exonuklease schneidet die immunpräzipierte DNA ein paar Base-Pairs (bp) von der Vernetzungsstelle, wodurch kontaminierende DNA abgebaut wird. Die exonukleasebehandelte ChIP-DNA wird aus den antikörperbeschichteten Perlen eluiert, Protein-DNA-Querverbindungen werden umgekehrt und Proteine abgebaut. DNA wird extrahiert und in einsträngige ChIP-DNA denaturiert, gefolgt von Primer-Glühen und Erweiterung, um doppelsträngige DNA (dsDNA) herzustellen. Als nächstes wird die Ligation eines Universaladapters an den exonukleasebehandelten Enden durchgeführt. Die resultierende DNA wird gereinigt, dann PCR verstärkt, gelgereinigt und der Sequenzierung der nächsten Generation unterzogen.
Das ChIP-exo-Protokoll ist länger als das ChIP-seq-Protokoll, aber technisch nicht sehr anspruchsvoll. Jede erfolgreich immunpräzipierte ChIP-DNA kann ChIP-exo mit mehreren zusätzlichen enzymatischen Schritten unterworfen werden. Die bemerkenswerten Vorteile von ChIP-exo, wie ultrahohe Mapping-Auflösung, ein reduziertes Hintergrundsignal und verringerte falsch positive und negative, in Bezug auf genomische Bindungsstellen, überwiegen die Zeitkosten.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >Agarose, UltraPure</td>
			<td >Invitrogen</td>
			<td >16500</td>
			<td >Checking Sonication (Section 4.3.6) and Gel Purification of LM-PCR Amplified DNA (Section 19.1)</td>
		</tr>
		<tr >
			<td >Albumin, Bovine Serum (BSA), Protease Free, Heat Shock Isolation, Min. 98%</td>
			<td >BioShop</td>
			<td >ALB003</td>
			<td >Blocking Solution</td>
		</tr>
		<tr >
			<td >Antibody against Isl1</td>
			<td >DSHB</td>
			<td >39.3F7</td>
			<td >Antibody incuation with beads (Section 5.6)</td>
		</tr>
		<tr >
			<td >Bioruptor Pico&#160;</td>
			<td >Diagenode</td>
			<td >B01060010</td>
			<td >Sonicating Chromatin (Section 3.3)</td>
		</tr>
		<tr >
			<td >Bovine serum albumin (BSA), Molecular Biology Grade</td>
			<td >New England BioLabs</td>
			<td >B9000S</td>
			<td >Fill-in Reaction on Beads (Section 10.2) and Denaturing, Primer Annealing and Primer Extension (Section 14.2)</td>
		</tr>
		<tr >
			<td >Centrifuge 5424 R</td>
			<td >Eppendorf</td>
			<td >5404000138</td>
			<td >Sonicating Chromatin (Section 3.5), Checking Sonication (Section 4.3.1, 4.3.3 and 4.3.4)</td>
		</tr>
		<tr >
			<td >Centrifuge 5804 R</td>
			<td >Eppendorf</td>
			<td >22623508</td>
			<td >Harvest, cross-linking and freezing cells (Section 1.3), Cell lysis (Section 2.2 and 2.3), Sonicating Chromatin (Section 3.1)</td>
		</tr>
		<tr >
			<td >cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail</td>
			<td >Roche</td>
			<td >4693159001</td>
			<td >Added to all buffers, except Proteinase K buffer and ChIP Elution buffer</td>
		</tr>
		<tr >
			<td >dATP Solution</td>
			<td >New England BioLabs</td>
			<td >N0440S</td>
			<td >dA-Tailing Reaction (Section 15.1)</td>
		</tr>
		<tr >
			<td >Deoxycholic Acid Sodium Salt</td>
			<td >fisher scientific</td>
			<td >BP349</td>
			<td >Lysis Buffer 3, High Salt Wash Buffer and LiCl Wash Buffer</td>
		</tr>
		<tr >
			<td >dNTP Mix, Molecular Biology Grade</td>
			<td >Thermo Scientific</td>
			<td >R0192</td>
			<td >Fill-in Reaction on Beads (Section 10.2) and Denaturing, Primer Annealing and Primer Extension (Section 14.2)</td>
		</tr>
		<tr >
			<td >DreamTaq Green PCR Master Mix, 2x&#160;</td>
			<td >Thermo Scientific</td>
			<td >K1081</td>
			<td >Ligation-Mediated PCR (Section 18.1)</td>
		</tr>
		<tr >
			<td >EDTA, 0.5 M, Sterile Solution, pH 8.0</td>
			<td >BioShop</td>
			<td >EDT111</td>
			<td >Lysis Buffer 1-3, Checking Sonication (Section 4.1), High Salt Wash Buffer, LiCl Wash Buffer and ChIP Elution Buffer</td>
		</tr>
		<tr >
			<td >Ethyl Alcohol Anhydrous, 100%</td>
			<td >Commercial alcohols</td>
			<td >P006EAAN</td>
			<td >Checking Sonication (Section 4.3.3)</td>
		</tr>
		<tr >
			<td >Formaldehyde, 36.5-38%, contains 10-15% methanol</td>
			<td >Sigma</td>
			<td >F8775</td>
			<td >Harvest, cross-linking and freezing cells (Section 1.1)</td>
		</tr>
		<tr >
			<td >Glycerol, Reagent Grade, min 99.5%</td>
			<td >BioShop</td>
			<td >GLY002</td>
			<td >Lysis Buffer 1</td>
		</tr>
		<tr >
			<td >Glycine, Biotechnology Grade, min. 99%</td>
			<td >BioShop</td>
			<td >GLN001</td>
			<td >Harvest, cross-linking and freezing cells (Section 1.2)</td>
		</tr>
		<tr >
			<td >Glycogen, RNA Grade</td>
			<td >Thermo Scientific</td>
			<td >R0551</td>
			<td >Checking Sonication (Section 4.3.3)</td>
		</tr>
		<tr >
			<td >HEPES, 1 M Sterile-filtered Solution, pH 7.3&#160;</td>
			<td >BioShop</td>
			<td >HEP003</td>
			<td >Lysis Buffer 1, High Salt Wash Buffer</td>
		</tr>
		<tr >
			<td >Klenow Fragment (3-&#62;5 exo-)</td>
			<td >New England BioLabs</td>
			<td >M0212S</td>
			<td >dA-Tailing Reaction (Section 15.1)</td>
		</tr>
		<tr >
			<td >Lambda exonuclease</td>
			<td >New England BioLabs</td>
			<td >M0262S</td>
			<td >Lambda Exonuclease Digestion on Beads (Section 11.2)</td>
		</tr>
		<tr >
			<td >Ligase Enhancer</td>
			<td >New England BioLabs</td>
			<td >E7645S</td>
			<td >NEBNext Ultra II DNA Library Kit. Index Adapter Ligation on Beads (Section 9.1) and Universal Adapter Ligation (Section 16.1)</td>
		</tr>
		<tr >
			<td >Ligase Master Mix</td>
			<td >New England BioLabs</td>
			<td >E7645S</td>
			<td >NEBNext Ultra II DNA Library Kit. Index Adapter Ligation on Beads (Section 9.1) and Universal Adapter Ligation (Section 16.1)</td>
		</tr>
		<tr >
			<td >Lithium Chloride (LiCl), Reagent grade</td>
			<td >Bioshop</td>
			<td >LIT704</td>
			<td >LiCl Wash Buffer</td>
		</tr>
		<tr >
			<td >Magnetic beads for ChIP (Dynabeads Protein G)</td>
			<td >Dynabeads Protein G (magnetic beads for ChIP)</td>
			<td >Dynabeads Protein G (magnetic beads for ChIP)</td>
			<td >Antibody incubation with beads (Section 5)</td>
		</tr>
		<tr >
			<td >Magnetic beads for DNA purification (AMPure XP Beads)</td>
			<td >Beckman Coulter</td>
			<td >A63880</td>
			<td >DNA Extraction (Section 13.3) and DNA Clean-up (Section 17.1)</td>
		</tr>
		<tr >
			<td >Magnetic rack (DynaMag-2 Magnet)</td>
			<td >Invitrogen</td>
			<td >12321D</td>
			<td >Used in many steps in Sections: 5 - 11, 13</td>
		</tr>
		<tr >
			<td >MinElute Gel Extraction Kit</td>
			<td >Qiagen&#160;</td>
			<td >28604</td>
			<td >Gel Purification of PCR Amplified DNA (Section 19.2)</td>
		</tr>
		<tr >
			<td >N-Lauroylsarcosine sodium salt solution, 30% aqueous solution, &#8805;97.0% (HPLC)</td>
			<td >Sigma</td>
			<td >61747</td>
			<td >Lysis Buffer 3</td>
		</tr>
		<tr >
			<td >Octylphenol Ethoxylate (IGEPAL CA630)</td>
			<td >BioShop</td>
			<td >NON999</td>
			<td >Lysis Buffer 1 and LiCl Wash Buffer</td>
		</tr>
		<tr >
			<td >Phenol:Chloroform:Isoamyl Alcohol, Biotechnology Grade (25:24:1)</td>
			<td >BioShop</td>
			<td >PHE512</td>
			<td >Checking Sonication (Section 4.3.1)</td>
		</tr>
		<tr >
			<td >phi29 DNA Polymerase</td>
			<td >New England BioLabs</td>
			<td >M0269L</td>
			<td >Fill-in Reaction on Beads (Section 10.2) and Denaturing, Primer Annealing and Primer Extension (Section 14.3 and 14.4)</td>
		</tr>
		<tr >
			<td >Phosphate-Buffered Saline (PBS), 1x&#160;</td>
			<td >Corning</td>
			<td >21040CV</td>
			<td >Harvest, cross-linking and freezing cells (Section 1.3) and Sonicating Chromatin (Section 3.1), Antibody incuation with beads (Section 5.1)</td>
		</tr>
		<tr >
			<td >PowerPac Basic Power Supply</td>
			<td >BioRad</td>
			<td >1645050</td>
			<td >Checking Sonication (Section 4.3.6) and Gel Purification of LM-PCR Amplified DNA (Section 19.1)</td>
		</tr>
		<tr >
			<td >ProFlex PCR System</td>
			<td >Applied Biosystems</td>
			<td >ProFlex PCR System</td>
			<td >Used in Sections: 14.2, 14.4, 15.2, 16.2 and 18.2</td>
		</tr>
		<tr >
			<td >Protein LoBind Tube, 2.0 mL&#160;</td>
			<td >Eppendorf</td>
			<td >22431102</td>
			<td >Antibody Incubation with Beads (Section 5.2) and Chromatin Immunoprecipitation (Section 6.3)</td>
		</tr>
		<tr >
			<td >Proteinase K Solution, RNA Grade</td>
			<td >Invitrogen</td>
			<td >25530049</td>
			<td >Checking Sonication (Section 4.2) and Elution and Reverse Crosslinking (Section 12.3)</td>
		</tr>
		<tr >
			<td >Qubit 4.0 Fluorometer</td>
			<td >Invitrogen</td>
			<td >Q33226</td>
			<td >Gel Purification of PCR Amplified DNA (Section 19.3)</td>
		</tr>
		<tr >
			<td >Quibit dsDNA BR assay kit</td>
			<td >Invitrogen</td>
			<td >Q32853</td>
			<td >Gel Purification of PCR Amplified DNA (Section 19.3)</td>
		</tr>
		<tr >
			<td >Rnase A, Dnase and Protease-free</td>
			<td >Thermo Scientific</td>
			<td >EN0531</td>
			<td >Checking Sonication (Section 4.3.2) and DNA Extraction (Section 13.2)</td>
		</tr>
		<tr >
			<td >Sodium chloride (NaCl), BioReagent&#160;</td>
			<td >Sigma</td>
			<td >S5886</td>
			<td >Lysis Buffer 1-3, High Salt Wash Buffer</td>
		</tr>
		<tr >
			<td >Sodium Dodecyl Sulfate (SDS), Electrophoresis Grade</td>
			<td >BioShop</td>
			<td >SDS001</td>
			<td >Checking Sonication (Section 4.1), High Salt Wash Buffer and ChIP Elution Buffer</td>
		</tr>
		<tr >
			<td >Sonication beads and 15 mL Bioruptor Tubes</td>
			<td >Diagenode</td>
			<td >C01020031</td>
			<td >Sonicating Chromatin (Section 3.1 and 3.2)</td>
		</tr>
		<tr >
			<td >ThermoMixer F1.5&#160;</td>
			<td >Eppendorf</td>
			<td >5384000020</td>
			<td >Section 4.2, 4.3.2, 4.3.5, 8.2, 9.2, 10.3, 11.2, 12.2, 12.3, 13.2 and 16.2</td>
		</tr>
		<tr >
			<td >Trizma hydrochloride solution (Tris-HCl), BioPerformance Certified, 1 M, pH 7.4</td>
			<td >Sigma</td>
			<td >T2194</td>
			<td >10 mM Tris-HCl Buffer</td>
		</tr>
		<tr >
			<td >Trizma hydrochloride solution (Tris-HCl), BioPerformance Certified, 1 M, pH 8.0</td>
			<td >Sigma</td>
			<td >T2694</td>
			<td >Lysis Buffer 2, Lysis Buffer 3, Checking Sonication (Section 4.1), LiCl Wash Buffer and ChIP Elution Buffer</td>
		</tr>
		<tr >
			<td >Ultra II End Repair/dA-Tailing Module (24 rxn -&#62; 120 rxn)</td>
			<td >New England BioLabs</td>
			<td >E7546S</td>
			<td >End Prep Reaction mix and End Prep Enzyme mix. End-repair and dA-Tailing Reaction on Beads (Section 8.2)</td>
		</tr>
		<tr >
			<td >VWR Mini Tube Rocker, Variable Speed</td>
			<td >VWR</td>
			<td >10159-752</td>
			<td >Used in many steps in sections: 1, 2, 5, 6 and 7</td>
		</tr>
		<tr >
			<td >2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol, Triton X-100, Reagent Grade</td>
			<td >BioShop</td>
			<td >TRX506</td>
			<td >Lysis Buffer 1, Sonicating Chromatin (Section 3.4) and High Salt Wash Buffer</td>
		</tr>
	</tbody>
</table>

high-resolution mapping of protein-dna interactions in mouse stem cell-derived neurons using chromatin immunoprecipitation-exonuclease (chip-exo)

Die Identifizierung spezifischer Protein-DNA-Wechselwirkungen im Genom ist wichtig für das Verständnis der Genregulation. Chromatin-Immunpräzipitation in Verbindung mit Hochdurchsatz-Sequenzierung (ChIP-seq) wird häufig verwendet, um genomweite Bindungsstellen von DNA-bindenden Proteinen zu identifizieren. Die ChIP-seq-Methode wird jedoch durch ihre Heterogenität in der Länge beschallter DNA-Fragmente und unspezifischer Hintergrund-DNA eingeschränkt, was zu einer geringen Kartierungsauflösung und Unsicherheit in DNA-bindenden Stellen führt. Um diese Einschränkungen zu überwinden, nutzt die Kombination von ChIP mit Exonuklease-Verdauung (ChIP-exo) die 5' bis 3' Exonuklease-Verdauung, um die heterogen große immunpräzipierte DNA auf die Protein-DNA-Vernetzungsstelle zu trimmen. Die Exonuklease-Behandlung eliminiert auch unspezifische Hintergrund-DNA. Die bibliotheksvorbereitete und exonukleaseverdsierte DNA kann zur Hochdurchsatzsequenzierung gesendet werden. Die ChIP-exo-Methode ermöglicht eine nahezu Basispaar-Mapping-Auflösung mit größerer Erkennungsempfindlichkeit und reduziertem Hintergrundsignal. Ein optimiertes ChIP-Exo-Protokoll für Säugetierzellen und die Sequenzierung der nächsten Generation wird im Folgenden beschrieben.

Abbildung 2A zeigt die Beschallungsergebnisse nach Zelllyse und Beschallung, mit verschiedenen Zyklen, von motorischen Neuronenzellen, die von Maus-ES-Zellen unterschieden werden. Die optimale Anzahl von Beschallungszyklen (z. B. 12 Zyklen in Abbildung 2A) erzeugte eine starke DNA-Intensität in DNA-Fragmenten von 100 bis 400 bp. Hochwertige ChIP-Exo-Bibliotheken basieren auf der Größe und Menge der fragmentierten Chromatin-DNA. Daher wird die Optimierung der ...

Watch this Scientific Journal Video about High-Resolution Mapping of Protein-DNA Interactions in Mouse Stem Cell-Derived Neurons using Chromatin Immunoprecipitation-Exonuclease ChIP-Exo at JoVE.com

High-Resolution Mapping of Protein-DNA Interactions in Mouse Stem Cell-Derived Neurons using Chromatin Immunoprecipitation-Exonuclease ChIP-Exo

Eine präzise Bestimmung von Proteinbindungsstellen im genomischen Ist wichtig für das Verständnis der Genregulation. Hier beschreiben wir eine genomische Kartierungsmethode, die chromatinimmunpräzipierte DNA mit Exonukleaseverdauung (ChIP-exo) behandelt, gefolgt von hochdurchsatzhoher Sequenzierung. Diese Methode erkennt Protein-DNA-Wechselwirkungen mit nahezu Base-Pair-Mapping-Auflösung und einem hohen Signal-Rausch-Verhältnis in Säugetierneuronen.

Hochauflösende Kartierung von Protein-DNA-Wechselwirkungen in Mausstammzell-abgeleiteten Neuronen mit Chromatin-Immunpräzipitation-Exonuklease (ChIP-Exo)

Identification of specific protein-DNA interactions on the genome is important for understanding gene regulation. Chromatin immunoprecipitation coupled ...

high-resolution-mapping-protein-dna-interactions-mouse-stem-cell

Research

JoVE Journal

Genetics

High-Resolution Mapping of Protein-DNA Interactions in Mouse Stem Cell-Derived Neurons using Chromatin Immunoprecipitation-Exonuclease (ChIP-Exo)

4.7K Aufrufe.  University of Toronto. Eine präzise Bestimmung von Proteinbindungsstellen im genomischen Ist wichtig für das Verständnis der Genregulation. Hier beschreiben wir eine genomische Kartierungsmethode, die chromatinimmunpräzipierte DNA mit Exonukleaseverdauung (ChIP-exo) behandelt, gefolgt von hochdurchsatzhoher Sequenzierung. Diese Methode erkennt Protein-DNA-Wechselwirkungen mit nahezu Base-Pair-Mapping-Auflösung und einem hohen Signal-Rausch-Verhältnis in Säugetierneuronen.

Serie: High-Resolution Mapping of Protein-DNA Interactions in Mouse Stem Cell-Derived Neurons using Chromatin Immunoprecipitation-Exonuclease ChIP-Exo

Mapping RNA-RNA Interactions Globally Using Biotinylated Psoralen

Knowing how RNAs interact with themselves and with others is key to understanding RNA based gene regulation in the cell. While examples of RNA-RNA interactions such as microRNA-mRNA interactions have been shown to regulate gene expression, the full extent to which RNA interactions occur in the cell is still unknown. Previous methods to study RNA interactions have primarily focused on subsets of RNAs that are interacting with a particular protein or RNA species. Here, we detail a method named Sequencing of Psoralen crosslinked, Ligated, and Selected Hybrids (SPLASH) that allows genome-wide capture of RNA interactions in vivo in an unbiased manner. SPLASH utilizes in vivo crosslinking, proximity ligation, and high throughput sequencing to identify intramolecular and intermolecular RNA base-pairing partners globally. SPLASH can be applied to different organisms including bacteria, yeast and human cells, as well as diverse cellular conditions to facilitate the understanding of the dynamics of RNA organization under diverse cellular contexts. The entire experimental SPLASH protocol takes about 5 days to complete and the computational workflow takes about 7 days to complete.

Here, we detail the method of Sequencing of Psoralen crosslinked, Ligated, and Selected Hybrids (SPLASH), which enables genome-wide mapping of intramolecular and intermolecular RNA-RNA interactions in vivo. SPLASH can be applied to study RNA interactomes of organisms including yeast, bacteria and humans.

Zuordnung von RNA-RNA-Wechselwirkungen unter Verwendung von biotinyliertem Psoralen

Knowing how RNAs interact with themselves and with others is key to understanding RNA based gene regulation in the cell. While examples of RNA-RNA ...

Forschung

Genetik

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

Genome-wide mapping of protein-DNA interactions is critical for understanding gene regulation, chromatin remodeling, and other chromatin-resident processes. Formaldehyde crosslinking followed by chromatin immunoprecipitation and high-throughput sequencing (X-ChIP-seq) has been used to gain many valuable insights into genome biology. However, X-ChIP-seq has notable limitations linked to crosslinking and sonication. Native ChIP avoids these drawbacks by omitting crosslinking, but often results in poor recovery of chromatin-bound proteins. In addition, all ChIP-based methods are subject to antibody quality considerations. Enzymatic methods for mapping protein-DNA interactions, which involve fusion of a protein of interest to a DNA-modifying enzyme, have also been used to map protein-DNA interactions. We recently combined one such method, chromatin endogenous cleavage (ChEC), with high-throughput sequencing as ChEC-seq. ChEC-seq relies on fusion of a chromatin-associated protein of interest to micrococcal nuclease (MNase) to generate targeted DNA cleavage in the presence of calcium in living cells. ChEC-seq is not based on immunoprecipitation and so circumvents potential concerns with crosslinking, sonication, chromatin solubilization, and antibody quality while providing high resolution mapping with minimal background signal. We envision that ChEC-seq will be a powerful counterpart to ChIP, providing an independent means by which to both validate ChIP-seq findings and discover new insights into genomic regulation.

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

We describe chromatin endogenous cleavage coupled with high-throughput sequencing (ChEC-seq), a chromatin immunoprecipitation (ChIP)-orthogonal method for mapping protein binding sites genome-wide with micrococcal nuclease (MNase) fusion proteins.

Genom-weite Abbildung von Protein-DNA-Wechselwirkungen mit ChEC-seq in<em&gt; Saccharomyces cerevisiae</em

Genome-wide mapping of protein-DNA interactions is critical for understanding gene regulation, chromatin remodeling, and other chromatin-resident ...

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 ...

Retroviral Scanning: Mapping MLV Integration Sites to Define Cell-specific Regulatory Regions

Moloney murine leukemia (MLV) virus-based retroviral vectors integrate predominantly in acetylated enhancers and promoters. For this reason, mLV integration sites can be used as functional markers of active regulatory elements. Here, we present a retroviral scanning tool, which allows the genome-wide identification of cell-specific enhancers and promoters. Briefly, the target cell population is transduced with an mLV-derived vector and genomic DNA is digested with a frequently cutting restriction enzyme. After ligation of genomic fragments with a compatible DNA linker, linker-mediated polymerase chain reaction (LM-PCR) allows the amplification of the virus-host genome junctions. Massive sequencing of the amplicons is used to define the mLV integration profile genome-wide. Finally, clusters of recurrent integrations are defined to identify cell-specific regulatory regions, responsible for the activation of cell-type specific transcriptional programs.
The retroviral scanning tool allows the genome-wide identification of cell-specific promoters and enhancers in prospectively isolated target cell populations. Notably, retroviral scanning represents an instrumental technique for the retrospective identification of rare populations (e.g. somatic stem cells) that lack robust markers for prospective isolation.

Here, we describe a protocol for genome-wide mapping of the integration sites of Moloney murine leukemia virus-based retroviral vectors in human cells.

Retroviral Scanning: Mapping MLV Integration Sites zur Definition zellspezifischer Regulierungsregionen

Moloney murine leukemia (MLV) virus-based retroviral vectors integrate predominantly in acetylated enhancers and promoters. For this reason, mLV ...

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

In our efforts to determine the patterns of expression and subcellular localization of Drosophila RNAs on a genome-wide basis, and in a variety of tissues, we have developed numerous modifications and improvements to our original fluorescent in situ hybridization (FISH) protocol. To facilitate throughput and cost effectiveness, all steps, from probe generation to signal detection, are performed using exon 96-well microtiter plates. Digoxygenin (DIG)-labelled antisense RNA probes are produced using either cDNA clones or genomic DNA as templates. After tissue fixation and permeabilization, probes are hybridized to transcripts of interest and then detected using a succession of anti-DIG antibody conjugated to biotin, streptavidin conjugated to horseradish peroxidase (HRP) and fluorescently conjugated tyramide, which in the presence of HRP, produces a highly reactive intermediate that binds to electron dense regions of immediately adjacent proteins. These amplification and localization steps produce a robust and highly localized signal that facilitates both cellular and subcellular transcript localization. The protocols provided have been optimized to produce highly specific signals in a variety of tissues and developmental stages. References are also provided for additional variations that allow the simultaneous detection of multiple transcripts, or transcripts and proteins, at the same time.

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

The described RNA in situ hybridization protocol allows the detection of RNA in whole Drosophila embryos or dissected tissues. Using 96-well microtiter plates and tyramide signal amplification, transcripts can be detected at high resolution, sensitivity, and throughput, and at a relatively low cost.

Hochauflösender Fluoreszenz In Situ Hybridisierung in Drosophila Embryos und Gewebe mit Tyramide Signalverstärkung

In our efforts to determine the patterns of expression and subcellular localization of Drosophila RNAs on a genome-wide basis, and in a variety of ...

Mapping Genome-wide Accessible Chromatin in Primary Human T Lymphocytes by ATAC-Seq

Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) is a method used for the identification of open (accessible) regions of chromatin. These regions represent regulatory DNA elements (e.g., promoters, enhancers, locus control regions, insulators) to which transcription factors bind. Mapping the accessible chromatin landscape is a powerful approach for uncovering active regulatory elements across the genome. This information serves as an unbiased approach for discovering the network of relevant transcription factors and mechanisms of chromatin structure that govern gene expression programs. ATAC-seq is a robust and sensitive alternative to DNase I hypersensitivity analysis coupled with next-generation sequencing (DNase-seq) and formaldehyde-assisted isolation of regulatory elements (FAIRE-seq) for genome-wide analysis of chromatin accessibility and to the sequencing of micrococcal nuclease-sensitive sites (MNase-seq) to determine nucleosome positioning. We present a detailed ATAC-seq protocol optimized for human primary immune cells i.e. CD4+ lymphocytes (T helper 1 (Th1) and Th2 cells). This comprehensive protocol begins with cell harvest, then describes the molecular procedure of chromatin tagmentation, sample preparation for next-generation sequencing, and also includes methods and considerations for the computational analyses used to interpret the results. Moreover, to save time and money, we introduced quality control measures to assess the ATAC-seq library prior to sequencing. Importantly, the principles presented in this protocol allow its adaptation to other human immune and non-immune primary cells and cell lines. These guidelines will also be useful for laboratories which are not proficient with next-generation sequencing methods.

Assay for Transposase-Accessible Chromatin coupled with high-throughput sequencing (ATAC-seq) is a genome-wide method to uncover accessible chromatin. This is a step-by-step ATAC-seq protocol, from molecular to the final computational analysis, optimized for human lymphocytes (Th1/Th2). This protocol can be adopted by researchers without prior experience in next-generation sequencing methods.

Mapping genomweite zugänglich Chromatin in primären menschlichen T-Lymphozyten durch ATAC-Seq

Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) is a method used for the identification of open (accessible) regions ...

Promoter Capture Hi-C: High-resolution, Genome-wide Profiling of Promoter Interactions

The three-dimensional organization of the genome is linked to its function. For example, regulatory elements such as transcriptional enhancers control the spatio-temporal expression of their target genes through physical contact, often bridging considerable (in some cases hundreds of kilobases) genomic distances and bypassing nearby genes. The human genome harbors an estimated one million enhancers, the vast majority of which have unknown gene targets. Assigning distal regulatory regions to their target genes is thus crucial to understand gene expression control. We developed Promoter Capture Hi-C (PCHi-C) to enable the genome-wide detection of distal promoter-interacting regions (PIRs), for all promoters in a single experiment. In PCHi-C, highly complex Hi-C libraries are specifically enriched for promoter sequences through in-solution hybrid selection with thousands of biotinylated RNA baits complementary to the ends of all promoter-containing restriction fragments. The aim is to then pull-down promoter sequences and their frequent interaction partners such as enhancers and other potential regulatory elements. After high-throughput paired-end sequencing, a statistical test is applied to each promoter-ligated restriction fragment to identify significant PIRs at the restriction fragment level. We have used PCHi-C to generate an atlas of long-range promoter interactions in dozens of human and mouse cell types. These promoter interactome maps have contributed to a greater understanding of mammalian gene expression control by assigning putative regulatory regions to their target genes and revealing preferential spatial promoter-promoter interaction networks. This information also has high relevance to understanding human genetic disease and the identification of potential disease genes, by linking non-coding disease-associated sequence variants in or near control sequences to their target genes.

DNA regulatory elements, such as enhancers, control gene expression by physically contacting target gene promoters, often through long-range chromosomal interactions spanning large genomic distances. Promoter Capture Hi-C (PCHi-C) identifies significant interactions between promoters and distal regions, enabling the assignment of potential regulatory sequences to their target genes.

Promotor Capture Hallo-c: Hochauflösende, genomweite Profilierung der Promoter Interaktionen

The three-dimensional organization of the genome is linked to its function. For example, regulatory elements such as transcriptional enhancers control the ...

CRISPR-Mediated Reorganization of Chromatin Loop Structure

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene expression, but whether looping is responsible for or a result of alterations in gene expression is still unknown. Until recently, how chromatin looping affects the regulation of gene activity and cellular function has been relatively ambiguous, and limitations in existing methods to manipulate these structures prevented in-depth exploration of these interactions. To resolve this uncertainty, we engineered a method for selective and reversible chromatin loop re-organization using CRISPR-dCas9 (CLOuD9). The dynamism of the CLOuD9 system has been demonstrated by successful localization of CLOuD9 constructs to target genomic loci to modulate local chromatin conformation. Importantly, the ability to reverse the induced contact and restore the endogenous chromatin conformation has also been confirmed. Modulation of gene expression with this method establishes the capacity to regulate cellular gene expression and underscores the great potential for applications of this technology in creating stable de novo chromatin loops that markedly affect gene expression in the contexts of cancer and development.

Chromatin looping plays a significant role in gene regulation; however, there have been no technological advances that allow for selective and reversible modification of chromatin loops. Here we describe a powerful system for chromatin loop re-organization using CRISPR-dCas9 (CLOuD9), demonstrated to selectively and reversibly modulate gene expression at targeted loci.

CRISPR-vermittelten Reorganisation der Chromatinstruktur mit Schleife

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene ...

High-Resolution Comparison of Bacterial Conjugation Frequencies

Bacterial conjugation is an important step in the horizontal transfer of antibiotic resistance genes via a conjugative DNA element. In-depth comparisons of conjugation frequency under different conditions are required to understand how the conjugative element spreads in nature. However, conventional methods for comparing conjugation frequency are not appropriate for in-depth comparisons because of the high background caused by the occurrence of additional conjugation events on the selective plate. We successfully reduced the background by introducing a most probable number (MPN) method and a higher concentration of antibiotics to prevent further conjugation in selective liquid medium. In addition, we developed a protocol for estimating the probability of how often donor cells initiate conjugation by sorting single donor cells into recipient pools by fluorescence-activated cell sorting (FACS). Using two plasmids, pBP136 and pCAR1, the differences in conjugation frequency in Pseudomonas putida cells could be detected in liquid medium at different stirring rates. The frequencies of conjugation initiation were higher for pBP136 than for pCAR1. Using these results, we can better understand the conjugation features in these two plasmids.

With an aim to understand the behaviors of various bacterial conjugative DNA elements under different conditions, we describe a protocol for detecting differences in conjugation frequency, with high resolution, to estimate how efficiently the donor bacterium initiates conjugation.

Hochauflösende Vergleich der bakteriellen Konjugation Frequenzen

Bacterial conjugation is an important step in the horizontal transfer of antibiotic resistance genes via a conjugative DNA element. In-depth comparisons ...

Chromatin Immunoprecipitation Assay Using Micrococcal Nucleases in Mammalian Cells

To express cellular phenotypes in organisms, living cells execute gene expression accordingly, and transcriptional programs play a central role in gene expression. The cellular transcriptional machinery and its chromatin modification proteins coordinate to regulate transcription. To analyze transcriptional regulation at the molecular level, several experimental methods such as electrophoretic mobility shift, transient reporter and chromatin immunoprecipitation (ChIP) assays are available. We describe a modified ChIP assay in detail in this article because of its advantages in directly showing histone modifications and the interactions between proteins and DNA in cells. One of the key steps in a successful ChIP assay is chromatin shearing. Although sonication is commonly used for shearing chromatin, it is difficult to identify reproducible conditions. Instead of shearing chromatin by sonication, we utilized enzymatic digestion with micrococcal nuclease (MNase) to obtain more reproducible results. In this article, we provide a straightforward ChIP assay protocol using MNase.

Chromatin immunoprecipitation (ChIP) is a powerful tool for understanding the molecular mechanisms of gene regulation. However, the method involves difficulties in obtaining reproducible chromatin fragmentation by mechanical shearing. Here, we provide an improved protocol for a ChIP assay using enzymatic digestion.

Chromatin Immunoprecipitation Assay mit Mikrokokken-Nukleasen in Säugetierzellen

To express cellular phenotypes in organisms, living cells execute gene expression accordingly, and transcriptional programs play a central role in gene ...