Name: the chip-exo method: identifying protein-dna interactions with near base pair precision
Uploaded: 2016-12-23T15:00:00
Description: Watch this Scientific Journal Video about The ChIP-exo Method: Identifying Protein-DNA Interactions with Near Base Pair Precision at JoVE.com

We thank the Venters Lab and members of the Molecular Physiology and Biophysics Department for helpful discussions. Special thanks to Frank Pugh for his guidance, mentoring, and many insightful discussions on the nuances of ChIP-exo while I was a post-doctoral fellow in his laboratory.

Hinweis: Doppelt destilliertes H 2 O oder UPW Äquivalent für alle Puffer und Reaktionen Mischungen empfohlen. Tag 0: Materialvorbereitung und Zellernte 1. Puffer Vorbereitung <ol><li> Bereiten Sie Lysepuffern 1-3 (Tabellen 1 - 3) und mit 100 &amp; mgr; l vollständige Protease - Inhibitor Lager (CPI) auf 50 ml jeder Puffer unmittelbar vor der Verwendung. Bereiten CPI Lager durch Auflösen einer Tablette in 1 ml UPW H 2 O. ...

<ol>
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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+an+Illumina-based+ChIP-exonuclease+method+provides+insight+into+FoxA1-DNA+binding+properties.">Development of an Illumina-based ChIP-exonuclease method provides insight into FoxA1-DNA binding properties.</a> Genome Biol. 14, R147 (2013).</li><li>He, Q., Johnston, J., Zeitlinger, 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-nexus+enables+improved+detection+of+in+vivo+transcription+factor+binding+footprints.">ChIP-nexus enables improved detection of in vivo transcription factor binding footprints.</a> Nat Biotechnol. 33, 395-401 (2015).</li></ol>

<html> Wir präsentieren eine funktionelle Genom Protokoll die genaue Bindungsstelle für Chromatin-interagierende Proteine ​​in einer unvoreingenommenen, genomweite Weise in der Nähe von Basenpaar-Auflösung zu bestimmen. Der wichtigste Schritt in der Nähe von Basenpaar Abbildungsauflösung zu erreichen, ist die Exonuclease-Behandlung des ChIP angereicherte DNA während der Immunopräzipitat auf dem magnetischen Harz bleibt. Vordergründig könnte Proteinkomplexe möglicherweise blockieren in vivo Fußabd...

Chromatinimmunpräzipitation (Chip-) ist eine leistungsfähige Methode Mechanismen der Genregulation zu untersuchen, indem selektiv für die DNA-Fragmente zu bereichern, die in lebenden Zellen mit einem bestimmten Protein in Wechselwirkung treten. Nachweisverfahren von ChIP angereicherte DNA - Fragmente haben sich weiterentwickelt , wie die Technologie verbessert, von der Erfassung eines einzelnen Locus (Standard ChIP-qPCR) zur Hybridisierung an Oligonukleotid - Mikroarrays (ChIP-Chip) auf Hochdurchsatz - Sequenzierung (ChIP-seq) 1. Obwohl ChIP-seq weit verbreitete Anwendung, Chromatin Heterogenität und unspezifischer DNA-Wechselwirkungen Datenqualität gesehen haben, was zu falsch-positive erschwert und ungenau Mapping. Um diese Einschränkungen zu umgehen, entwickelte Dr. Frank Pugh dem Chip-exo - Methode 2. Das hervorstechendste Merkmal von ChIP-exo ist, dass es eine 5 &#39;zu 3&#39; Exonuklease enthält, effektiv Transkriptionsfaktor-Bindungsstellen Footprinting. Im Ergebnis erreicht der Chip-exo-Methodik höhere Auflösung, größeren Dynamikbereich von detection und unteren Hintergrundgeräusche. Obwohl ChIP-exo technisch schwer zu meistern ist als ChIP-seq ist es weithin angenommen wird , wie Studien einzigartige , ultrahochauflösende Einblicke mit verschiedenen biologischen Systemen 3-8 gewinnen wollen. Tatsächlich ChIP-exo wurde auf Bakterien, Hefe, Maus, Ratte und menschlichen Zellsysteme erfolgreich eingesetzt. Als Beweis für das Prinzip wurde ChIP-exo ursprünglich verwendet , um die genaue Bindungsmotiv für eine Handvoll von Hefe - Transkriptions zu identifizieren 2 Faktoren ab . Die Technik wurde auch in Hefe verwendet , um die Organisation des Transkriptions Präinitiationskomplex zu studieren, und subnucleosomal Struktur verschiedener Histone 9,10 zu dechiffrieren. In jüngerer Zeit, Leveraged wir ChIP-exo benachbarten TFIIB und Pol II an menschlichen Promotoren Bindungsereignisse zu lösen, und zeigte , dass weit verbreitete divergente Transkription entsteht aus verschiedenen Initiations 11 - Komplexe. Der Workflow hier präsentiert wird optimiert und streamlined für Säugetier ChIP-exo (Abbildung 1). Erstens leben primäre oder Gewebekulturzellen mit Formaldehyd behandelt in vivo Protein-DNA - Wechselwirkungen durch eine kovalente Vernetzungs zu erhalten. Die Zellen werden lysiert und Chromatin abgeschert ~ 100-500 Basenpaar Größe Fragmente. ChIP dann reichert selektiv für DNA an das Protein von Interesse vernetzt Fragmente. An diesem Punkt ChIP-seq-Bibliotheken werden typischerweise hergestellt, die von Natur aus begrenzt die Detektionsauflösung auf die durchschnittliche Fragmentgröße von wenigen hundert Basenpaaren. Jedoch ChIP-exo überwindet diese Einschränkung durch die linken und rechten 5&#39;-DNA Grenzen der Protein-DNA-Vernetzungsstelle mit Lambda-Exonuklease Trimmen. Sequencing Bibliotheken werden dann aus Exonuklease verdaut DNA konstruiert, wie unten beschrieben. Die resultierenden verschachtelten 5 &#39;Grenzen stellen eine in vivo Footprint des Protein-DNA - Interaktion (Abbildung 1, Schritt 14) und werden durch Hochdurchsatz - Sequenzierung nachgewiesen. Although der Chip-exo-Methodik mehr beteiligt als ChIP-seq ist, Übergänge zwischen den meisten Schritten erfordert einfache Perle Waschen, die Probenverlust und experimentelle Variabilität minimiert. Wichtig ist, dass da ChIP-exo ist eine verfeinerte Version von ChIP-seq, jede Probe, die mit ChIP-seq sollte auch erfolgreich sein, mit ChIP-exo erfolgreich ist. Die in - vivo - Fußabdrucks von Protein-DNA - Wechselwirkungen mit ChIP-exo Ergebnisse in einer grundlegend unterschiedliche Datenstruktur von ChIP-seq. Obwohl häufig vorkommend ChIP-seq Anrufern ChIP-exo-Daten angewendet werden kann, die präzisesten Spitzen Anrufe zu erhalten, empfehlen wir die Bioinformatik Tools, die speziell mit der einzigartigen Struktur von ChIP-exo-Daten im Auge behalten. Dazu gehören Genetrack, GEM, MACE, Peakzilla und ExoProfiler 12-15.

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			<td >37% formaldehyde, methanol free, 10x10ml ampules</td>
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			<td >Complete Protease Inhibitor cocktail (CPI)</td>
			<td >Roche Life Science</td>
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			<td >Section 5</td>
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			<td >15 ml polystyrene tubes</td>
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			<td >MagSepharose Protein G Xtra beads</td>
			<td >GE Healthcare</td>
			<td >28-9670-66</td>
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			<td >DynaMag-1.5ml Side Magnetic Rack</td>
			<td >Invitrogen</td>
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			<td >Mini-Tube Rotator</td>
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			<td >DNA Polymerase I, Klenow</td>
			<td >New England BioLabs</td>
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			<td >10x NEBuffer 2 (10x Reaction Buffer 2)</td>
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			<td >ATP</td>
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			<td >T4 Polynucleotide Kinase</td>
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			<td >dATP</td>
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			<td >Klenow 3&#39;-5&#39; Exo Minus</td>
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			<td >T4 DNA ligase&#160;</td>
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			<td >10x &#934;-29 Buffer</td>
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			<td >New England BioLabs</td>
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			<td >10x Lambda Buffer</td>
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			<td >New England BioLabs</td>
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			<td >Proteinase K</td>
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			<td >Glycogen</td>
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			<td >10x T4 Ligase Buffer</td>
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			<td >AMPure XP (clean up) beads&#160;</td>
			<td >Beckman Coulter</td>
			<td >A63881</td>
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			<td >Q5 Hot Start DNA Polymerase&#160;</td>
			<td >New England BioLabs</td>
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			<td >5x Q5 Buffer (5x PCR Buffer)</td>
			<td >New England BioLabs</td>
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			<td >Qubit Fluorometer</td>
			<td >Invitrogen</td>
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			<td >QIAquick Gel Extraction Kit</td>
			<td >Qiagen</td>
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			<td >Optical Clear Qubit tubes&#160;</td>
			<td >Invitrogen</td>
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			<td >Section 26</td>
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			<td >Qubit dsDNA High Sensitivity Assay kit</td>
			<td >Invitrogen</td>
			<td >Q32851</td>
			<td >Section 26</td>
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the chip-exo method: identifying protein-dna interactions with near base pair precision

Chromatinimmunpräzipitation (Chip-) ist ein unverzichtbares Werkzeug in den Bereichen Epigenetik und Genregulation, die spezifische Protein-DNA-Wechselwirkungen isoliert. ChIP gekoppelt Hochdurchsatz-Sequenzierung (ChIP-seq) wird allgemein die genomische Lage von Proteinen zu bestimmen, die mit Chromatin in Wechselwirkung treten. Allerdings ChIP-seq wird durch eine relativ geringe Abbildungsauflösung von mehreren hundert Basenpaaren und hohen Hintergrundsignal behindert. Der Chip-exo-Methode ist eine verfeinerte Version von ChIP-seq, die auf sowohl Auflösung und Rauschen wesentlich verbessert. Der Hauptunterschied der ChIP-exo-Methodik ist die Einarbeitung von lambda-Exonuclease-Verdauung in der Bibliothek Vorbereitungsarbeitsablauf, um effektiv die linke Standfläche und rechts 5 &#39;DNA-Grenzen des Protein-DNA-Vernetzungsstelle. Der Chip-exo-Bibliotheken werden dann einer Hochdurchsatz-Sequenzierung unterzogen. Die resultierenden Daten können genutzt werden, um eine einzigartige und extrem hochauflösende Einblicke in die funktionelle Organisation of das Genom. Hier beschreiben wir die ChIP-exo-Methode, die wir optimiert und rationalisiert für Säugetier-Systeme der nächsten Generation Sequenzierung-durch-Synthese-Plattform.

Die folgenden Abbildungen zeigen repräsentative Ergebnisse aus dem Chip-exo-Protokoll hier vorgestellt. Im Gegensatz zu herkömmlichen ChIP-seq Methodologien mit wenigen enzymatischen Schritten, ChIP-exo erfordert elf sequentiell abhängigen enzymatischen Reaktionen (Abbildung 1). Somit muss darauf geachtet werden bei jedem Schritt unternommen werden, um sicherzustellen, dass jede Reaktionskomponente seiner jeweiligen Reaktions Mastermix zugesetzt wird. Wir empfehlen, e...

Watch this Scientific Journal Video about The ChIP-exo Method: Identifying Protein-DNA Interactions with Near Base Pair Precision at JoVE.com

The ChIP-exo Method: Identifying Protein-DNA Interactions with Near Base Pair Precision

Here, we present a protocol to achieve near base pair resolution of protein-DNA interactions. This is obtained by exonuclease treatment of DNA fragments selectively enriched by chromatin immunoprecipitation (ChIP-exo) followed by high throughput sequencing.

Der Chip-exo-Methode: Ermittlung von Protein-DNA-Wechselwirkungen mit Near-Basenpaar Precision

Chromatin immunoprecipitation (ChIP) is an indispensable tool in the fields of epigenetics and gene regulation that isolates specific protein-DNA ...

The overall goal of this functional genomic technology is to identify the precise genomic locations that a given protein occupies in living cells. This method can help answer key questions in the fields of gene regulation and epigenetics such as how the transcription machinery assembles and operates in the context of chromatin. The main advantage of this technique is it reveals the exact genomic location for protein DNA interactions at near-base pre-resolution with exceptionally low background.Generally, individuals new to this method will struggle because the protocol is complex and requires attention to detail throughout. To sonicate nuclear lysates, begin by placing resin adapters in 15 milliliter polystyrene tubes containing nuclear extracts. Sonicate the lysates in an ice cold water bath at medium power for 30 seconds on and 30 seconds off for a total of two 15-minute sessions.To check sonication results, transfer two 10 microliter samples from each tube into 1.5 milliliter tubes. Then reverse crosslinks by combining the first 10 microliter sample with 10 microliters of TE-RNase A and 0.2 microliters of proteinase K.Incubate the tube at 37 degrees Celsius for 30 minutes. Then add four microliters of 6X xylene DNA dye.To the second 10 microliter sample, preserve crosslinks by adding two microliters of 6X xylene DNA dye. Run both samples on a 1.5%agarose gel at 140 volts for approximately 30 to 45 minutes until the bromophenol dye in the latter is three-quarters of the way down the gel. If sonication was successful, transfer sonicated lysates to two milliliter tubes containing 150 microliters of 10%Triton X-100 and vortex to mix.Pellet the insoluble chromatin and debris by spinning the sonicated lysates at 20, 000 times g at four degrees Celsius for 10 minutes. Then transfer the supernatants to new two milliliter tubes. Store the samples at negative 80 degrees Celsius indefinitely.For each chromatin immunoprecipitation or ChIP reaction, combine 1.5 milliliters of sonicated extracts with antibody bead conjugates in 1.5 milliliter tubes. Incubate the tubes on a mini tube rotator at a speed of nine at four degrees Celsius overnight. Check after a few minutes to ensure samples are not leaking and are mixing properly.The following day, very briefly spin down the tubes to collect the liquid in the caps and place the tubes on a magnetic rack for one minute. Then aspirate the extract. Add 0.75 milliliters of RIPA buffer to each tube.Then remove the tubes from the magnetic rack and invert them several times to mix. After a brief spin, place the tubes back on the magnet and aspirate the supernatant. Following the last wash, add 0.75 milliliters of 10 millimolar Tris HCL to each tube.Remove the tubes from the magnetic rack and invert the samples several times to mix. After a brief spin, place the tubes back on the rack and aspirate the supernatant. After carrying out the polishing and detailing reactions according to the text protocol, fill out the ligation master mix calculations.Then make the ligation mix in a 1.5 milliliter tube on ice and pipette to mix the solution. With the sample tubes on the magnetic rack, add 48 microliters of P7 ligation master mix and two microliters of a different adapter index to each of the sample resins. Incubate the samples in a ThermoMixer at three times g in 25 degrees Celsius for two hours.After washing the samples and performing the Phy 29 nick repair and kinase reactions according to the text protocol, fill out the lambda exonuclease master mix calculations and make the lambda exonuclease mix in a 1.5 milliliter tube on ice. Add 50 microliters of lambda exonuclease mix to each sample resin while still on the magnetic rack. Then incubate the samples in a ThermoMixer at three times g in 37 degrees Celsius for 30 minutes.After aspirating the last wash as described in the text protocol, carry out RecJf nuclease reaction by filling out the RecJf master mix calculations and making the RecJf mix on ice. Add 50 microliters of the RecJf mix to each sample resin on the magnetic rack. Then incubate the samples in a ThermoMixer at three times g in 37 degrees Celsius for 30 minutes.After aspirating the last wash, prepare the ChIP elution buffer and proteinase K master mix and add 200 microliters of the solution to each sample resin. Incubate the samples in a ThermoMixer at three times g in 65 degrees Celsius overnight with a heated lid to prevent condensation. The following day, briefly spin the samples and place the tubes on the magnetic rack for one minute.Then transfer 200 microliters of supernatant to a new 1.5 milliliter tube containing 200 microliters of TE buffer. Following DNA extraction, P7 primary extension and ATL reactions, fill out the P5 adapter ligation table master mix calculations and make the ligation master mix on ice. Add 20 microliters of the ligation mix to each sample and pipette to mix.Then incubate the samples in a thermal cycler at 25 degrees Celsius for two hours. Carry out DNA purification and quantification according to the text protocol. As shown here, it is worth spending time to optimize sonication conditions to obtain high-quality chromatin extracts that yield DNA fragments between 100 to 500 base pairs.And electrophoretic mobility shift assay is used to verify that the sonicated extracts contain intact protein DNA crosslinks, evident as a super shift of DNA fragments. To assess background signal, a mock ChIP is routinely performed without antibody in the reaction. A high-quality ChIP exo-library will yield very little background in the mock sample compared to a ChIP-seq library preparation.Shown here is a bioanalyzer assessment of a ChIP-exo library. Analysis accurately ensures the library size distribution and detects contaminating adapter dimers that run at 125 base pairs. This panel illustrates the smooth distribution of strand-separated ChIP-exo tag five prime Ns for polymerase II, TFIIB and TBP at the human RPS12 gene in proliferating K562 cells.Shown in this figure are average ChIP-exo patterns relative to the transcriptional start sites of protein coding genes. The major peak locations for TBP, TFIIB and polymerase II in the composite plot reflect the genomic organization of human transcription initiation complexes. Once mastered, this technique can be done in four days if performed properly.After its development, this technique paved the way for researchers in the field of gene regulation to explore molecular details of subnuclear structure in assymetry in yeast. Following this procedure, other methods like RNase seek expression profiling can be performed to address questions such as whether a given factor influences the expression of a nearby gene. While attempting this procedure, it's important to remember to carefully fill out master mix tables for all enzymatic steps.Don't forget, working with carcinogens and other caustic agents can be extremely hazardous and precautions such as wearing personal protective equipment should always be taken while performing this procedure. After this watching video, you should have a good understanding of how to prepare a high-quality ChIP-exo library.

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