Name: high-resolution mapping of protein-dna interactions in mouse stem cell-derived neurons using chromatin immunoprecipitation-exonuclease (chip-exo)
Uploaded: 2020-08-14T16:00:00
Description: 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

Vi takker medlemmet af Rhee-laboratoriet for at dele ikke-offentliggjorte data og værdifulde diskussioner. Dette arbejde blev støttet af Natural Sciences and Engineering Research Council of Canada (NSERC) tilskud RGPIN-2018-06404 (HR).

BEMÆRK: Autoklaveret destilleret og deioniseret vand (ddH2O) anbefales til fremstilling af buffere og reaktionsmasterblandinger. Afsnit 1−4 beskriver celle lysis og sonikering, afsnit 5−7 beskriver chromatin immunoprecipitation (ChIP), afsnit 8−11 beskriver enzymatiske reaktioner på perler, afsnit 12 og 13 beskriver ChIP elution og DNA-rensning, og afsnit 14−19 beskriver biblioteksforberedelse.
1. Høst og krydsning af celler
<ol><li>Efter at have differ...

<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 Biology. 14 (12), 147 (2013).</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=Comprehensive+genome-wide+protein-DNA+interactions+detected+at+single-nucleotide+resolution.">Comprehensive genome-wide protein-DNA interactions detected at single-nucleotide resolution.</a> Cell. 147 (6), 1408-1419 (2011).</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> Current Protocols in Molecular Biology. Chapter 21, 21-24 (2012).</li><li>Starick, S. 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=ChIP-exo+signal+associated+with+DNA-binding+motifs+provides+insight+into+the+genomic+binding+of+the+glucocorticoid+receptor+and+cooperating+transcription+factors.">ChIP-exo signal associated with DNA-binding motifs provides insight into the genomic binding of the glucocorticoid receptor and cooperating transcription factors.</a> Genome Research. 25 (6), 825-835 (2015).</li><li>Han, G. 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. F., Yan, Q., Wang, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deciphering+the+regulon+of+a+GntR+family+regulator+via+transcriptome+and+ChIP-exo+analyses+and+its+contribution+to+virulence+in+Xanthomonas+citri.">Deciphering the regulon of a GntR family regulator via transcriptome and ChIP-exo analyses and its contribution to virulence in Xanthomonas citri.</a> Molecular Plant Pathology. 18 (2), 249-262 (2017).</li><li>Kim, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systems+assessment+of+transcriptional+regulation+on+central+carbon+metabolism+by+Cra+and+CRP.">Systems assessment of transcriptional regulation on central carbon metabolism by Cra and CRP.</a> Nucleic Acids Research. 46 (6), 2901-2917 (2018).</li><li>Niu, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+genome-wide+binding+interactions+of+mouse+and+human+constitutive+androstane+receptors+reveal+novel+gene+targets.">In vivo genome-wide binding interactions of mouse and human constitutive androstane receptors reveal novel gene targets.</a> Nucleic Acids Research. 46 (16), 8385-8403 (2018).</li><li>Uuskula-Reimand, 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=Topoisomerase+II+beta+interacts+with+cohesin+and+CTCF+at+topological+domain+borders.">Topoisomerase II beta interacts with cohesin and CTCF at topological domain borders.</a> Genome Biology. 17, (2016).</li><li>Rhee, H. 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. 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I denne protokol bruges ChIP efterfulgt af exonuklease fordøjelse til at opnå DNA-biblioteker til identifikation af protein-DNA-interaktioner i pattedyrsceller ved ultrahøj kortlægningsopløsning. Mange variabler bidrager til kvaliteten af ChIP-exo eksperimentet. Kritiske eksperimentelle parametre omfatter kvaliteten af antistoffer, optimering af sonikering og antallet af LM-PCR-cyklusser. Disse kritiske eksperimentelle parametre er også, hvad der kan begrænse ChIP-exo eksperimenter og vil blive diskuteret nedenfor...

Placeringen af protein-DNA-interaktioner giver indsigt i mekanismerne for genregulering. Chromatin immunoprecipitation kombineret med høj gennemstrømningssekvensering (ChIP-seq) har været brugt i et årti til at undersøge genom-dækkende protein-DNA-interaktioner i levende celler1,2. ChIP-seq-metoden er imidlertid begrænset af heterogenitet i DNA-fragmentering og ubundet DNA-forurening, der fører til lav kortlægningsopløsning, falske positiver, ubesvarede opkald og ikke-specifikt baggrundssignal. Kombinationen af ChIP med exonuklease fordøjelse (ChIP-exo) forbedrer ChIP-seq-metoden ved at trimme ChIP DNA til protein-DNA crosslinking-punkterne, hvilket giver nær base-pair-opløsning og et lavt baggrundssignal3,4,5. Den stærkt forbedrede kortlægningsopløsning og lave baggrund fra ChIP-exo gør det muligt at bestemme nøjagtige og omfattende protein-DNA-bindingssteder på tværs af genomet. ChIP-exo er i stand til at afsløre funktionelt forskellige DNA-bindende motiver, samarbejdsinteraktioner mellem transskriptionsfaktorer (TF) og flere protein-DNA-krydslinkingsteder på et bestemt genomisk bindingssted, der ikke kan påvises ved andre genomiske kortlægningsmetoder3,4,6,7.
ChIP-exo blev oprindeligt brugt i spirende gær til at undersøge den sekvensspecifikke DNA-binding af TF'er, til at studere den nøjagtige organisering af transskriptionsforstartskomplekset og subkernestrukturen af individuelle histoner på tværs af genomet4,8,9. Siden introduktionen i 20114er ChIP-exo med succes blevet udnyttet i mange andre organismer, herunder bakterier, mus og menneskelige celler7,10,11,12,13,14,15,16,17. I 2016 brugte Rhee et al.14 ChIP-exo i pattedyrsneuroner for første gang for at forstå, hvordan neuronalt genekspression blev opretholdt efter nedreguleringen af programmeringen TF Lhx3, som danner et heterodimer kompleks med en anden programmering TF Isl1. Denne undersøgelse viste, at Isl1 i mangel af Lhx3 rekrutteres til nye neuronale forstærkere bundet af Onecut1 TF for at opretholde genekspression af neuronale effektorgener. I denne undersøgelse afslørede ChIP-exo, hvordan flere TF'er dynamisk genkender celletype og cellestadiespecifikke DNA-reguleringselementer på en kombinatorisk måde ved næsten nukleotidkortopløsning. Andre undersøgelser brugte også ChIP-exo-metoden til at forstå samspillet mellem proteiner og DNA i andre pattedyrscellelinjer. Han et al.7 brugte ChIP-exo til at undersøge genom-dækkende organisering af GATA1 og TAL1 TF'er i mus erythroid celler ved hjælp af ChIP-exo. Denne undersøgelse viste, at TAL1 er direkte rekrutteret til DNA snarere end indirekte gennem protein-protein interaktioner med GATA1 hele erythroid differentiering. Nylige undersøgelser også brugt ChIP-exo til at profilere genom-dækkende bindende steder CTCF, RNA Polymerase II, og histone mærker til at studere epigenomiske og transskriptionelle mekanismer i menneskelige cellelinjer18,19.
Der findes flere versioner af ChIP-exo-protokollen3,5,20. Disse ChIP-exo-protokoller er dog vanskelige at følge for forskere, der ikke er bekendt med næste generations sekventeringsbiblioteksforberedelse. En fremragende version af ChIP-exo-protokollen blev offentliggjort med let at følge instruktioner og en video21, men indeholdt mange enzymatiske trin, der kræver en betydelig mængde tid at gennemføre. Her rapporterer vi en ny version af ChIP-exo-protokollen, der indeholder reducerede enzymatiske trin og inkubationstider og forklaringer til hvert enzymatisk trin21. Slutreparations- og dA-tailing-reaktioner kombineres i et enkelt trin ved hjælp af endeforberedelsesenzym. Inkubationstiden for indeks- og universaladapterens ligationstrin reduceres fra 2 timer til 15 min. ved hjælp af en ligationforstærker. Kinasereaktionen efter det ligationstrin for indeksadapteren, der er beskrevet i den forrige ChIP-exo-protokol, fjernes. I stedet tilsættes en fosfatgruppe under oligo-DNA-syntesen til en af indeksadapterens 5 ender (tabel 1), som vil blive brugt til lambda exonuklease fordøjelsestrinnet. Mens den tidligere ChIP-exo-protokol brugte RecJf exonuclease fordøjelsen til at eliminere enkeltstrengede DNA-forurenende stoffer, fjernes dette fordøjelsestrin her, fordi det ikke er kritisk for kvaliteten af ChIP-exo-biblioteket. For at rense omvendt krydslinket DNA efter ChIP-udsmeltning anvendes desuden en magnetisk perlerensningsmetode i stedet for ekstraktionsmetoden phenol:chloroform:isoamylalkohol (PCIA). Dette reducerer inkubationstiden for DNA-ekstraktion. Det er vigtigt, at det fjerner de fleste adapter dimers dannet under indeksadapter ligation, hvilket kan påvirke effektiviteten af ligation-medieret PCR.
ChIP-exo-protokollen, der præsenteres her, er optimeret til påvisning af præcise protein-DNA-interaktioner i pattedyrsneuroner, der er differentieret fra musefosterstamceller (ES). Kort, høstede og crosslinked neuronale celler lysed, at tillade chromatin at blive udsat for sonikering, derefter sonikeret således, at passende størrelse DNA-fragmenter er opnået (Figur 1). Antistofbelagte perler anvendes derefter til selektivt at immunoprecipitate fragmenteret, opløselig kromatin til protein af interesse. Mens det immunoprecipiterede DNA stadig er på perlerne, udføres endereparation, ligation af sekventeringsadaptere, udfyldningsreaktion og 5' til 3' lambda exonuclease fordøjelsestrin. Exonuklease fordøjelsestrinnet er det, der giver ChIP-exo sin ultrahøje opløsning og høje signal-til-støj-forhold. Lambda exonuclease trimmer immunoprecipiteret DNA et par base-par (bp) fra crosslinking site, hvilket forårsager forurenende DNA, der skal nedbrydes. Det exonukleasebehandlede ChIP DNA er udvisket fra de antistofbelagte perler, protein-DNA crosslinks vendes, og proteiner nedbrydes. DNA udvindes og denatureres til enkeltstrenget ChIP DNA, efterfulgt af primer annealing og udvidelse til at lave dobbeltstrenget DNA (dsDNA). Dernæst udføres ligation af en universel adapter til de exonukleasebehandlede ender. Den resulterende DNA renses, derefter PCR forstærkes, gel renset, og udsættes for næste generation sekventering.
ChIP-exo-protokollen er længere end ChIP-seq-protokollen, men er ikke særlig teknisk udfordrende. Enhver vellykket immunoprecipiteret ChIP DNA kan udsættes for ChIP-exo, med flere yderligere enzymatiske trin. De bemærkelsesværdige fordele ved ChIP-exo, såsom ultrahøj kortlægningsopløsning, et reduceret baggrundssignal og reducerede falske positive og negativer med hensyn til genomiske bindingssteder, opvejer tidsomkostningerne.

<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)

Identifikation af specifikke protein-DNA-interaktioner på genomet er vigtig for forståelsen af genregulering. Chromatin immunoprecipitation kombineret med høj-gennemløb sekventering (ChIP-seq) er meget udbredt til at identificere genom-dækkende bindende steder af DNA-bindende proteiner. ChIP-seq-metoden er imidlertid begrænset af dens heterogenitet i længden af sonikerede DNA-fragmenter og ikke-specifik baggrunds-DNA, hvilket resulterer i lav kortlægningsopløsning og usikkerhed på DNA-bindingssteder. For at overvinde disse begrænsninger bruger kombinationen af ChIP med exonuklease fordøjelse (ChIP-exo) 5' til 3'exonuklease fordøjelse til at trimme det heterogene størrelse immunoprecipiterede DNA til protein-DNA crosslinking-stedet. Exonuklease behandling eliminerer også ikke-specifik baggrunds-DNA. Det biblioteksforberedte og eksorklease fordøjede DNA kan sendes til sekvensering med høj gennemløb. ChIP-exo-metoden giver mulighed for næsten basispartilknytningsopløsning med større registreringsfølsomhed og reduceret baggrundssignal. En optimeret ChIP-exo-protokol til pattedyrsceller og næste generations sekventering er beskrevet nedenfor.

Figur 2A illustrerer sonikeringsresultater efter celle lysis og sonikering, med forskellige cyklusser, af motoriske neuronceller differentieret fra mus ES celler. Det optimale antal sonikeringscyklusser (f.eks. 12 cyklusser i figur 2A) genererede stærk DNA-intensitet i 100−400 BP DNA-fragmenter. ChIP-exo-biblioteker af høj kvalitet er baseret på størrelsen og mængden af fragmenteret chromatin-DNA. Således anbefales optimering af sonikering for hver celle...

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

Præcis bestemmelse af proteinbindende steder på tværs af genomet er vigtig for forståelsen af genregulering. Her beskriver vi en genomisk kortlægningsmetode, der behandler chromatin-immunoprecipitated DNA med exonuklease fordøjelse (ChIP-exo) efterfulgt af højgennemstrømning sekventering. Denne metode registrerer protein-DNA-interaktioner med nær base-pair kortlægning opløsning og høj signal-til-støj-forholdet i pattedyr neuroner.

Kortlægning i høj opløsning af protein-DNA-interaktioner i mus stamcelle-afledte neuroner ved hjælp af Chromatin Immunoprecipitation-Exonuclease (ChIP-Exo)

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

The main advantage of our ChIP-exo method is the improved mapping resolution of protein DNA interactions in the cell compared to the conventional chromatin immunoprecipitation method. To prepare protein G magnetic beads for ChIP, mix the magnetic beads until homogeneous. Then add 25 microliters of the magnetic beads to a two milliliter protein low bind tube.Add one milliliter of blocking solution to the beads. Mix well by pipetting, then place the tube on a magnetic rack for one minute. Once the supernatant is clear, remove it.Next, add one milliliter of blocking solution to the beads. Place the tube on a rocking platform at four degrees Celsius and rock for 10 minutes, then briefly spin the tube. Place the tube on the magnetic rack and remove the supernatant.Add 500 microliters of blocking solution to the magnetic beads. Briefly spin the antibody against Islet-1, then add four micrograms of the antibody to the tube containing the magnetic beads. Place the tube containing magnetic beads and Islet-1 antibody on a rocking platform at four degrees Celsius and rock for six to 24 hours.To begin the chromatin immunoprecipitation, wash the antibody-coated beads in one milliliter of blocking solution. Place the tube containing the antibody-coated beads on a rocking platform at four degrees Celsius for five minutes. After briefly spinning, place the tube on a magnetic rack and remove the supernatant.Resuspend the antibody-coated beads in 50 microliters of blocking solution. Add one milliliter of sonicated lysates to the antibody-coated beads, then incubate the sample on a rocking platform at four degrees Celsius overnight. After allowing the sample to incubate overnight, collect liquid from the cap by briefly spinning the tube, then place the tube on a magnetic rack.And after one minute, remove the supernatant carefully with a pipette. Next, perform a series of washes as follows. Add one milliliter of cold wash buffer containing CPI to the sample.Mix the sample on a rocking platform at four degrees Celsius for five minutes. Briefly spin the sample tube, then place on a magnetic rack. After one minute, remove the supernatant with a pipette.For the first enzymatic reaction and repair and DA tailing, add 38 microliters of autoclaved double distilled water to the sample, then add end prep reaction mix and end prep enzyme mix. Mix the contents of the tube by pipetting, then incubate the sample at 20 degrees Celsius for 30 minutes. Next, wash the sample beads with high salt wash buffer, lithium chloride wash buffer, and 10 millimolar Tris hydrogen chloride buffer as described previously.To perform the index adapter ligation, add 27 microliters of cold 10 millimolar Tris hydrogen chloride buffer to the sample. Then add index adapter, ligation enhancer, and ligase master mix. Incubate the sample at 20 degrees Celsius for 15 minutes.Again, wash the sample beads with high salt wash buffer, lithium chloride wash buffer, and 10 millimolar Tris hydrogen chloride buffer. Then add 47 microliters of cold 10 millimolar Tris hydrogen chloride buffer to the sample. To repair the nick in the DNA resulting from the missing phosphodiester bond, add 11.1 microliters of the fill-in mix to the sample.Incubate the sample at 30 degrees Celsius for 20 minutes. After a series of washes, add 50 microliters of cold autoclaved double distilled water to the sample. To digest the ChIP DNA in the five prime to three prime direction, add lambda exonuclease buffer and lambda exonuclease and mix the sample by pipetting.Incubate the sample at 37 degrees Celsius for 30 minutes. After the digestion is complete, repeat the three washes a final time. To elute ChIP sample from the beads, resuspend the sample in 75 microliters of ChIP elution buffer and incubator at 65 degrees Celsius for 15 minutes at 130 times G.Add 2.5 microliters of 20 milligrams per milliliter proteinase K to the sample.Vortex the sample briefly, then incubate overnight at 65 degrees Celsius. Briefly spin the sample and place on a magnetic rack. After one minute, transfer the supernatant to a new 1.5 milliliter tube.Purify and elute the DNA. After purifying the eluted DNA, transfer 16 microliters of the extracted DNA sample to a PCR tube. Add 1.2 microliters of denaturing and primer annealing mix to the sample.Using the program described in table six of the manuscript, denature and anneal primers to the template DNA. Add three microliters of primer extension mix to the sample and run the PCR program described in table seven of the manuscript. Add 4.1 microliters of DA tailing mix to the sample and run the sample using the PCR program in table eight of the manuscript.Then add 21.5 microliters of universal adapter ligation mix to the sample and incubate for 15 minutes at 20 degrees Celsius. Add 29 microliters of LM-PCR mix to the sample, then run the sample using the program from table 10 of the manuscript. Following 18 cycles of LM-PCR, ChIP-exo samples from mouse motor neurons were electrophoresed on a 1.5%agarose gel.The results for ChIP-exo Islet-1 showed amplified DNA libraries around 200 to 400 base pairs indicating that the ChIP-exo libraries were successfully amplified by LM-PCR. The band around 100 base pairs represents artifacts from adapters and PCR primers. The no antibody control demonstrates that non-specific background DNA was digested by the lambda exonuclease treatment.Using ChIP-exo and ChIP-seq, Islet-1 bound locations were identified. The ChIP-exo signal was highly focused at Islet-1 binding sites detecting multiple clustered Islet-1 transcription factor binding patterns. The ChIP-seq signal displayed broader signals indicating that ChIP-exo had higher mapping resolution than ChIP-seq for Islet-1.Multiple transcription factors often bind DNA next to each other as a protein-DNA complex. ChIP-exo allows us to study how multiple transcription factors recognize and regulate target DNA.

Research

JoVE Journal

Genetics

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

Kortlægning af RNA-RNA-interaktioner globalt ved anvendelse af biotinyleret 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 ...

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

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

Genom-bred kortlægning af protein-DNA-interaktioner med 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

Chromatin Immunoprecipitation ChIP in Mouse T-cell Lines

Chromatin-immunpræcipitation (ChIP) i mus-T-cellelinier

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

Retroviral Scanning: Kortlægning af MLV Integration Sites til at definere Cell-specifikke Regulatory Regions

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

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

Høj opløsning fluorescerende In Situ hybridisering i Drosophila fostre og væv ved hjælp af Tyramide Signal forstærkning

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

Kortlægning Genome-wide tilgængelige kromatin i primære humane T lymfocytter af 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

Promotor Capture Hej-C: Høj opløsning, Genome-wide profilering af promotor interaktioner

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

CRISPR-medieret reorganisering af kromatin løkke struktur

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

Høj opløsning sammenligning af bakteriel konjugation frekvenser

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

Kromatin Immunoprecipitation assay ved hjælp af Mikrococcal-nukleaser i pattedyrsceller

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