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.

Not: Çift distile H 2 O veya moleküler sınıf eşdeğer tüm tamponlar ve reaksiyonlar karışımları için tavsiye edilir. Gün 0: Malzeme Hazırlama ve Hücre Hasat 1. Tampon Hazırlama <ol><li> 3 (Tablo 1 - 3 -) Lizis Tampon 1 hazırlanması ve kullanımdan hemen önce, her tampon, 50 ml 100 ul tam proteaz inhibitör stokları (CPI) ekleyin. Bir tablet 1 mL moleküler sınıf H 2 O eriterek TÜFE stok hazırlayın </l...

<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. F., 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=Identification+of+transcription+factor+binding+sites+from+ChIP-seq+data+at+high+resolution.">Identification of transcription factor binding sites from ChIP-seq data at high resolution.</a> Bioinformatics. 29, 2705-2713 (2013).</li><li>Wang, L., 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=MACE:+model+based+analysis+of+ChIP-exo.">MACE: model based analysis of ChIP-exo.</a> Nucleic Acids Res. 42, e156 (2014).</li><li>Bentley, D. 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=Accurate+whole+human+genome+sequencing+using+reversible+terminator+chemistry.">Accurate whole human genome sequencing using reversible terminator chemistry.</a> Nature. 456, 53-59 (2008).</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=ChIP-exo+method+for+identifying+genomic+location+of+DNA-binding+proteins+with+near-single-nucleotide+accuracy.">ChIP-exo method for identifying genomic location of DNA-binding proteins with near-single-nucleotide accuracy.</a> Curr Protoc Mol Biol. Chapter 21, Unit 21.24 (2012).</li><li>Landt, S. 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. 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<html> Biz kromatin yakın baz çifti çözünürlükte bir tarafsız, genom şekilde proteinlerin etkileşim için kesin bağlayıcı konumunu belirlemek için fonksiyonel genomik protokol mevcut. İmmüno manyetik reçine kalırken yakın baz çifti haritalama çözünürlük elde etmek için en kritik adım ChIP zenginleştirilmiş DNA eksonükleaz tedavi yöntemidir. Görünüşte, protein kompleksleri potansiyel herhangi bir alt birimi (örn kromatin remodeling kompleksleri veya nükleozom çekirdek parçac...

Kromatin immünopresipitasyon (ChIP) seçici canlı hücreler belirli bir protein ile etkileşime DNA fragmanlarının için zenginleştirerek gen regülasyonu mekanizmaları incelemek için güçlü bir yöntemdir. Teknolojisi, yüksek verimlilik sıralaması (ChIP-seq) 1 ila oligonükleotid array (ChIP-chip) üzerine melezleşme tek bir lokus (standart ChIP-qPCR) tespiti itibaren, iyileştikçe ChIP zenginleştirilmiş DNA parçalarının tespit yöntemleri geliştirmişlerdir. ChIP-seq yaygın bir uygulama, kromatin heterojeniteyi ve nonspesifik DNA etkileşimleri gördü rağmen yanlış pozitif ve kesin olmayan eşleme önde gelen veri kalitesi engel olmuştur. Bu sınırlamaları aşmak için, Dr. Frank Pugh ChIP-ekzo yöntemi 2 geliştirdi. ChIP-ekso belirgin özelliği etkin yerleri bağlayıcı transkripsiyon faktörü ayak izi, ekzonükleaz &#39;ila 3&#39; 5 sahip olmasıdır. Sonuç olarak, ChIP-exo metodolojisi daha yüksek çözünürlük, gözetleme daha dinamik aralık elden ve alt arka plan gürültü. ChIP-exo daha teknik açıdan zor ChIP-seq daha usta olmasına rağmen çalışmalar çeşitli biyolojik sistemler 3-8 kullanarak benzersiz ultra yüksek çözünürlüklü anlayışlar kazanmak amacı olarak, yaygın olarak kabul ediliyor. Gerçekten de, yonga-ekso başarılı bakteri, maya, fare, sıçan ve insan hücre sistemlerine uygulanmıştır. Prensip kanıtı olarak, ChIP-exo başlangıçta maya transkripsiyon bir avuç 2 faktörleri için kesin bağlayıcı motifi tanımlamak için kullanıldı. Bu teknik aynı zamanda, transkripsiyon, ön başlatma kompleksinin organizasyonunu çalışma ve çeşitli histon 9,10 subnucleosomal yapısını çözmek için maya kullanılmıştır. Daha yakın zamanlarda, insan rehberleri etkinliklerde bağlama bitişik TFIIB ve Pol II gidermek için ChIP-exo kaldıraçlı ve farklı başlangıç 11 kompleksleri gelen yaygın farklı transkripsiyon ortaya gösterdi. Burada sunulan iş akışı optimize ve sMemeli ChIP-ekso (Şekil 1) için treamlined. İlk olarak, canlı birincil ya da doku kültürü hücreleri, bir kovalent çapraz bağlanırlar in vivo protein-DNA etkileşim bölgesindeki korumak için formaldehit ile muamele edilir. Hücreler parçalanır ve kromatin ~ 100 makaslanmış - 500 baz çifti boyutu fragmanları. ChIP sonra seçici ilgili protein çapraz bağlanmış DNA parçaları için zenginleştirir. Bu noktada, yonga-DİZİ kütüphaneler doğal birkaç yüz baz çifti ortalama fragman büyüklüğü algılama çözünürlüğünü sınırlar hazırlanır. Ancak, ChIP-exo sol ve lambda eksonükleaz ile protein-DNA çapraz sitenin sağ 5 &#39;DNA sınırları kırparak bu sınırlama üstesinden gelir. Aşağıda ayrıntılı olarak açıklandığı gibi Ardışık kütüphaneleri eksonükleaz ile sindirilmiş DNA inşa edilir. Elde edilen iç içe geçmiş 5 &#39;sınırları protein-DNA etkileşim (Şekil 1, aşama 14), bir in vivo ayak izi temsil eder ve yüksek sekanslama ile tespit edilir. Although ChIP-ekzo metodolojisi ChIP-seq daha katılır, çoğu adımlar arasındaki geçişler örnek kaybı ve deneysel değişkenliği en aza indirir basit boncuk yıkama gerektirir. Önemlisi, ChIP-ekzo ChIP-seq, aynı zamanda ChIP-exo başarılı olmalıdır ChIP-seq ile başarılı herhangi bir örnek bir rafine versiyonu beri. ChIP-seq bir temelde farklı bir veri yapısında ChIP-ekzo sonuçları ile protein-DNA etkileşimleri in vivo ayak izi. Ortak ChIP-seq arayanlar en hassas zirve aramaları elde etmek için, ChIP-ekzo verilerine tatbik edilebilir ancak biz özellikle akılda ChIP-ekzo verilerin benzersiz yapısı ile tasarlanmış biyoinformatik araçlar öneriyoruz. Bunlar Genetrack, GEM, OKVO, Peakzilla ve ExoProfiler 12-15 arasındadır.

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

Kromatin immünopresipitasyon (ChIP) epigenetik ve spesifik protein-DNA etkileşimlerini izolatlarının gen regülasyonu alanlarında vazgeçilmez bir araçtır. Yüksek verimli sekanslama (Chip seq) bağlı ChIP yaygın kromatin etkileşim proteinlerin genomik konumunu belirlemek için kullanılır. Ancak, ChIP-seq birkaç yüz baz çifti nispeten düşük haritalama çözünürlük ve yüksek arka plan sinyali tarafından engellenmektedir. ChIP-ekzo yöntemi esas çözünürlük ve gürültü hem geliştirir ChIP-seq bir rafine versiyonu. ChIP-ekzo metodolojisinin kilit ayrım etkili bir sol ayak izini kütüphane hazırlık iş akışında lambda eksonükleaz sindirim birleşme ve protein-DNA çapraz sitenin sağ 5 &#39;DNA sınırları olduğunu. ChIP-ekso diziler daha sonra, yüksek sekanslama tabi tutulur. Elde edilen veriler fonksiyonel organizasyon o içine eşsiz ve ultra-yüksek çözünürlüklü bir anlayış sağlamak için kaldıraçlı olabilirgenom f. Burada, biz optimize edilmiş ve memeli sistemlerinde ve yeni nesil dizileme-by-sentez platformu için aerodinamik olan ChIP-exo yöntem açıklanmaktadır.

Aşağıdaki şekiller Burada sunulan ChIP-exo protokol temsili sonuçlar göstermektedir. Birkaç enzimatik adımda bilinen bir çipin seq metodolojilere aksine, yonga-ekso on ardışık göre enzimatik reaksiyonları (Şekil 1) gerekmektedir. Bu nedenle, bakım, her reaksiyon bileşeni, ilgili reaksiyon ana karışıma ilave edilir sağlamak için her adımda alınmalıdır. Biz ortaya çıkan tablo baskı, otomatik olarak reaksiyon master miks hesaplamaları gerçekle...

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.

ChIP-ekzo Yöntem: Base Çifti Hassas Protein-DNA Etkileşimleri belirlenmesi

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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