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.

Nota: Doble H2O destilada o equivalente grado molecular se recomienda para todos los tampones y las reacciones mezclas. Día 0: Preparación del material y de las Pilas de cosecha 1. tampón de preparación de <ol><li> Preparar lisis Buffers 1 - 3 (Tablas 1 - 3) y añadir 100 l de inhibidor de proteasa completo de la (CPI) a 50 ml de cada tampón justo antes del uso. Preparar IPC de stock de disolución de un comprimido en 1 ml de grad...

<ol>
	<li>Venters, B. J., 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=How+eukaryotic+genes+are+transcribed.">How eukaryotic genes are transcribed.</a> Crit Rev Biochem Mol Biol. 44, 117-141 (2009).</li><li>Rhee, H. S., Pugh, B. 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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, 295-301 (2012).</li><li>Rhee, H. S., Bataille, A. R., Zhang, L., 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. , 1377-1388 (2014).</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, e0149339 (2016).</li><li>Albert, I., Wachi, S., Jiang, C., 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=GeneTrack--a+genomic+data+processing+and+visualization+framework.">GeneTrack--a genomic data processing and visualization framework.</a> Bioinformatics. 24, 1305-1306 (2008).</li><li>Guo, Y., Mahony, S., Gifford, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+resolution+genome+wide+binding+event+finding+and+motif+discovery+reveals+transcription+factor+spatial+binding+constraints.">High resolution genome wide binding event finding and motif discovery reveals transcription factor spatial binding constraints.</a> PLoS computational biology. 8, e1002638 (2012).</li><li>Bardet, A. 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G., 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=ChIP-seq+guidelines+and+practices+of+the+ENCODE+and+modENCODE+consortia.">ChIP-seq guidelines and practices of the ENCODE and modENCODE consortia.</a> Genome Res. 22, 1813-1831 (2012).</li><li>Serandour, A. A., Brown, G. D., Cohen, J. D., Carroll, J. 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> Se presenta un protocolo de genómica funcional para determinar la ubicación exacta de unión de la cromatina proteínas que interactúan de una manera imparcial, en todo el genoma a una resolución de pares de bases próximo. El paso más crítico para lograr la base cerca de una resolución de mapeo par es el tratamiento con exonucleasa de la ADN-chip enriquecido mientras que el inmunoprecipitado permanece en la resina magnética. Aparentemente, los complejos de proteínas potencialmente podrían bloquear la hu...

inmunoprecipitación de la cromatina (ChIP) es un método potente para estudiar los mecanismos de la regulación de genes por enriquecer selectivamente para fragmentos de ADN que interactúan con una proteína dada en las células vivas. Los métodos de detección de fragmentos de ADN chip enriquecida han evolucionado a medida que la tecnología mejora, desde la detección de un solo locus (estándar chip-qPCR) para la hibridación de microarrays de ADN (chip-chip) a secuenciación de alto rendimiento (chip-ss) 1. Aunque el chip-ss ha visto amplia aplicación, la heterogeneidad de la cromatina y las interacciones no específicas de ADN han obstaculizado calidad de los datos que conduce a falsos positivos y la cartografía imprecisa. Para sortear estas limitaciones, el Dr. Frank Pugh desarrolló el método de chip-exo 2. La característica sobresaliente de chip-exo es que incorpora un 5 &#39;a 3&#39; exonucleasa, footprinting efectivamente factor de transcripción sitios de unión. Como resultado, la metodología chip-exo logra una resolución más alta, mayor rango dinámico de detection, y un menor ruido de fondo. Aunque el chip-exo es técnicamente más difícil de dominar que el chip-ss, que está siendo ampliamente adoptado como estudios apuntan a obtener una visión única de ultra alta resolución utilizando diversos sistemas biológicos 3-8. De hecho, el chip-exo se ha aplicado con éxito a bacterias, levaduras, ratón, rata, y los sistemas de células humanas. Como prueba de principio, chip-exo fue originalmente utilizado para identificar el motivo de unión precisa para un puñado de la transcripción de levadura Factores 2. La técnica también se utilizó en la levadura para estudiar la organización del complejo de pre-iniciación de la transcripción, y para descifrar estructura subnucleosomal de diversas histonas 9,10. Más recientemente, hemos aprovechado chip-exo para resolver TFIIB adyacente y Pol II promotores de eventos en humanos vinculante, y demostramos que surge de la transcripción divergente generalizada de los distintos complejos de iniciación 11. El flujo de trabajo que aquí se presenta se optimiza y streamlined de mamíferos chip-exo (Figura 1). En primer lugar, las células vivas de cultivo primario o de tejidos se tratan con formaldehído para preservar en las interacciones proteína-ADN in vivo a través de una reticulación covalente. Las células se lisan y la cromatina esquiladas a ~ 100 - 500 pares de bases de tamaño de los fragmentos. ChIP entonces enriquece selectivamente fragmentos de ADN para reticular para la proteína de interés. En este punto, las bibliotecas ChIP-seq son típicamente preparados, lo que limita inherentemente la resolución de detección para el tamaño medio de fragmento de unos pocos cientos de pares de bases. Sin embargo, el chip-exo supera esta limitación por el recorte de la derecha 5 fronteras &#39;de ADN del sitio de entrecruzamiento de proteínas de ADN con exonucleasa lambda e izquierdo. bibliotecas de secuenciación entonces se construyen a partir de ADN digerido exonucleasa como se detalla a continuación. Los anidados 5 &#39;fronteras resultantes representan una huella in vivo de la interacción proteína-ADN (Figura 1, paso 14), y se detectan mediante secuenciación de alto rendimiento. altHough la metodología chip-exo es más complicado que el chip-ss, las transiciones entre la mayoría de los pasos requiere el lavado del grano simple, lo que minimiza la pérdida de muestra y la variabilidad experimental. Es importante destacar que, desde el chip-exo es una versión refinada de chip-ss, cualquier muestra que tiene éxito con chip-ss también debe tener éxito con chip-exo. La huella in vivo de las interacciones proteína-ADN con resultados chip-exo en una estructura de datos fundamentalmente distinto del chip-ss. A pesar de que las personas que llaman comunes chip-ss pueden aplicarse a los datos de chip-exo, para obtener las llamadas pico más precisas se recomienda la bioinformática herramientas diseñadas específicamente con la estructura única de datos chip-exo en mente. Estos incluyen Genex, GEM, MACE, Peakzilla, y ExoProfiler 12-15.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >37% formaldehyde, methanol free, 10x10ml ampules</td>
			<td >ThermoFisher Scientific</td>
			<td >28908</td>
			<td >Section 3</td>
		</tr>
		<tr >
			<td >Complete Protease Inhibitor cocktail (CPI)</td>
			<td >Roche Life Science</td>
			<td >11873580001</td>
			<td >Sections 4, 8</td>
		</tr>
		<tr >
			<td >Bioruptor sonicator</td>
			<td >Diagenode</td>
			<td >UCD200</td>
			<td >Section 5</td>
		</tr>
		<tr >
			<td >15 ml polystyrene tubes</td>
			<td >BD Falcon</td>
			<td >352095</td>
			<td >Section 5</td>
		</tr>
		<tr >
			<td >MagSepharose Protein G Xtra beads</td>
			<td >GE Healthcare</td>
			<td >28-9670-66</td>
			<td >Section 6</td>
		</tr>
		<tr >
			<td >DynaMag-1.5ml Side Magnetic Rack</td>
			<td >Invitrogen</td>
			<td >12321D</td>
			<td >Sections 6-17, 22</td>
		</tr>
		<tr >
			<td >Mini-Tube Rotator</td>
			<td >Fisher Scientific</td>
			<td >05-450-127</td>
			<td >Sections 7, 22</td>
		</tr>
		<tr >
			<td >T4 DNA Polymerase</td>
			<td >New England BioLabs</td>
			<td >M0203</td>
			<td >Section 9</td>
		</tr>
		<tr >
			<td >DNA Polymerase I, Klenow</td>
			<td >New England BioLabs</td>
			<td >M0210</td>
			<td >Section 9</td>
		</tr>
		<tr >
			<td >10x NEBuffer 2 (10x Reaction Buffer 2)</td>
			<td >New England BioLabs</td>
			<td >B7002</td>
			<td >Sections 9-11, 13, 15, 20</td>
		</tr>
		<tr >
			<td >Thermomixer C</td>
			<td >Eppendorf</td>
			<td >5382</td>
			<td >Sections 9-16</td>
		</tr>
		<tr >
			<td >ATP</td>
			<td >Roche Life Science</td>
			<td >010419979001</td>
			<td >Sections 9, 11, 13</td>
		</tr>
		<tr >
			<td >dNTPs</td>
			<td >New England BioLabs</td>
			<td >N0447</td>
			<td >Sections 9, 12, 19, 23</td>
		</tr>
		<tr >
			<td >T4 Polynucleotide Kinase</td>
			<td >New England BioLabs</td>
			<td >M0201</td>
			<td >Sections 9, 13</td>
		</tr>
		<tr >
			<td >dATP</td>
			<td >New England BioLabs</td>
			<td >N0440</td>
			<td >Sections 10, 20</td>
		</tr>
		<tr >
			<td >Klenow 3&#39;-5&#39; Exo Minus</td>
			<td >New England BioLabs</td>
			<td >M0212</td>
			<td >Sections 10, 20</td>
		</tr>
		<tr >
			<td >T4 DNA ligase&#160;</td>
			<td >New England BioLabs</td>
			<td >M0202</td>
			<td >Sections 11, 21</td>
		</tr>
		<tr >
			<td >&#934;-29 DNA Polymerase</td>
			<td >New England BioLabs</td>
			<td >M0269</td>
			<td >Sections 12, 19</td>
		</tr>
		<tr >
			<td >10x &#934;-29 Buffer</td>
			<td >New England BioLabs</td>
			<td >B0269</td>
			<td >Sections 12, 19</td>
		</tr>
		<tr >
			<td >Lambda Exonuclease</td>
			<td >New England BioLabs</td>
			<td >M0262</td>
			<td >Section 14</td>
		</tr>
		<tr >
			<td >10x Lambda Buffer</td>
			<td >New England BioLabs</td>
			<td >B0262</td>
			<td >Section 14</td>
		</tr>
		<tr >
			<td >RecJf Exonuclease</td>
			<td >New England BioLabs</td>
			<td >M0264</td>
			<td >Section 15</td>
		</tr>
		<tr >
			<td >Proteinase K</td>
			<td >Roche Life Science</td>
			<td >03115828001</td>
			<td >Section 16</td>
		</tr>
		<tr >
			<td >Glycogen</td>
			<td >Roche Life Science</td>
			<td >010901393001</td>
			<td >Section 18</td>
		</tr>
		<tr >
			<td >10x T4 Ligase Buffer</td>
			<td >New England BioLabs</td>
			<td >B0202</td>
			<td >Section 21</td>
		</tr>
		<tr >
			<td >AMPure XP (clean up) beads&#160;</td>
			<td >Beckman Coulter</td>
			<td >A63881</td>
			<td >Section 22</td>
		</tr>
		<tr >
			<td >Q5 Hot Start DNA Polymerase&#160;</td>
			<td >New England BioLabs</td>
			<td >M0493</td>
			<td >Section 23</td>
		</tr>
		<tr >
			<td >5x Q5 Buffer (5x PCR Buffer)</td>
			<td >New England BioLabs</td>
			<td >B9027</td>
			<td >Section 23</td>
		</tr>
		<tr >
			<td >Qubit Fluorometer</td>
			<td >Invitrogen</td>
			<td >Q33216</td>
			<td >Section 25</td>
		</tr>
		<tr >
			<td >QIAquick Gel Extraction Kit</td>
			<td >Qiagen</td>
			<td >28704</td>
			<td >Section 25</td>
		</tr>
		<tr >
			<td >Optical Clear Qubit tubes&#160;</td>
			<td >Invitrogen</td>
			<td >Q32856</td>
			<td >Section 26</td>
		</tr>
		<tr >
			<td >Qubit dsDNA High Sensitivity Assay kit</td>
			<td >Invitrogen</td>
			<td >Q32851</td>
			<td >Section 26</td>
		</tr>
	</tbody>
</table>

the chip-exo method: identifying protein-dna interactions with near base pair precision

inmunoprecipitación de la cromatina (CHIP) es una herramienta indispensable en el campo de la epigenética y la regulación de genes específicos que aísla las interacciones proteína-ADN. ChIP acoplado a la secuenciación de alto rendimiento de (ChIP-seq) se usa comúnmente para determinar la localización genómica de las proteínas que interactúan con la cromatina. Sin embargo, el chip-ss se ve obstaculizada por una resolución relativamente baja mapeo de varios cientos de pares de bases y alta señal de fondo. El método de chip-exo es una versión refinada de chip-ss que mejora sustancialmente cuando se produzca tanto la resolución y el ruido. La distinción clave de la metodología chip-exo es la incorporación de la digestión de exonucleasa lambda en el flujo de trabajo de preparación de la biblioteca a la huella efectiva la izquierda y derecha 5 fronteras &#39;de ADN del sitio de entrecruzamiento de proteínas de ADN. Las bibliotecas de chip-exo se someten a secuenciación de alto rendimiento. Los datos resultantes se puede aprovechar para proporcionar una visión única y ultra-alta resolución en la organización funcional of genoma. A continuación, se describe el método de chip-exo que hemos optimizado y simplificado para los sistemas de mamíferos y de próxima generación de plataformas de secuenciación por síntesis.

Las siguientes figuras ilustran los resultados representativos del chip de protocolo-exo que aquí se presenta. En contraste con las metodologías tradicionales de chip-ss con pocos pasos enzimáticos, chip-exo requiere once reacciones enzimáticas secuencialmente dependientes (Figura 1). Por lo tanto, se debe tener cuidado en cada paso para asegurarse de que cada componente de reacción se agrega a su respectiva mezcla maestra de reacción. Recomendamos la generación d...

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.

El Método chip-exo: La identificación de las interacciones proteína-ADN con zona de Base Par de precisión

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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