Wir bedanken uns bei allen Mitarbeitern des IGBMC Hochdurchsatz-Sequenzierung Anlage, ein Mitglied des Konsortiums "Frankreich-Génomique" (ANR10-INBS-09-08) und alle IGBMC allgemeine Dienstleistungen insbesondere das Personal der IGBMC Tierhaltung. Diese Arbeit wurde unterstützt durch Zuschüsse aus dem CNRS, INSERM, AFM, der Ligue Nationale Contre le Cancer, die französische staatliche Fonds durch die ANR im Rahmen des Programms Investissements Avenir gekennzeichnet ANR-10-IDEX-0002-02, die Labex INRT ANR-10-IDEX-0001-02 und die ANR-AR2GR-16-CE11-009-01. Die Geldgeber hatten keine Rolle beim Studiendesign, Datenerhebung und Analyse, Entscheidung, zu veröffentlichen oder der Manuskripterstellung. S.J wurde unterstützt von der Ligue Nationale Contre le Cancer und die ANR-AR2GR-16-CE11-009-01 und V.U durch das Ministère de nannte et De La Recherche. ID ist ein 'Équipe Labellisée' der Ligue Nationale Contre le Cancer.

Mäuse gehalten wurden, im Einklang mit den institutionellen Richtlinien zur Pflege und Verwendung von Labortieren und nationale Tier-Pflege-Richtlinien (Europäische Kommission Richtlinie 86/609/EWG; Französischen Dekret Nr. 87-848). Alle Verfahren wurden von der französischen nationalen Ethikkommission genehmigt.
 1. Isolation von Muskelgewebe <ol><li>Opfer eines Erwachsenen 6 bis 8 Wochen altes Baby Maus durch zervikale Dislokation. Sterlise der Extremität durch Spülen mit 70 % Ethanol und sezieren die hinteren Gliedmaßen Muskeln (Gastrocnemius, m. Tibialis Anterior, Quadrizeps) mit feinen Spitze Schere und Zange. 
	Hinweis: Sollte eine Maus etwa 500 mg des Gewebes ergeben.</li>
	<li>Hackfleisch die Muskeln in einem 2 mL Reagenzglas mit 1 mL eiskaltes hypotone Puffer, um eine homogene Zubereitung von kleinen (&#60; ca. 2-3 mm 3) Stücke mit feinen Schere und verlassen das Rohr schütteln auf einer Bank Top Rührwerk bei 4 ° C für ca. 5-10 min.</li>
</ol> 2. Gewebe-Lyse Hinweis: siehe Tabelle 1 für alle Kompositionen Puffern.
<ol><li>Das Homogenat in einem 14 mL Rundboden Röhrchen zu übertragen und in 5 mL kalte hypotone Puffer (EDTA-freie Protease-Inhibitor cocktail und PMSF) Aufschwemmen.
	<ol><li>Homogenise das Hackfleisch / Muskelgewebe mit einer lockeren Dounce (20-30 Schläge innerhalb von 3 Minuten) oder eine mechanische Gewebe-Homogenisator für 15-30 s in 14 mL Rundboden Röhrchen. 
		Hinweis: Die Effizienz der Lyse in diesem Stadium durch Lichtmikroskopie beurteilt werden kann und bei Bedarf zusätzliche Störungen durchgeführt werden kann.</li>
	</ol></li> <li>Das Homogenat in 15 mL-Tuben und Lautstärke auf 10 mL mit kalten hypotone Puffer zu füllen. Das Homogenat zu beheben, wie unten beschrieben, bevor Sie mit der nächsten Maus.</li>
</ol> 3. Befestigung <ol><li>1 % Endkonzentration Formaldehyd hinzu und schütteln Sie für 10 min bei Raumtemperatur.</li>
	<li>In jedem Röhrchen, Fixierung und schütteln für 5-10 min bei Raumtemperatur zu stoppen eine Endkonzentration von 0,125 M Glycin hinzufügen.</li>
</ol> 4. Kerne Vorbereitung Hinweis: siehe Tabelle 1 für alle Kompositionen Puffern.
<ol><li>Homogenise die feste lysate mit einem losen Dounce (5-10 Schläge).
	<ol><li>Übertragung eine 15 mL Tube und Zentrifuge bei 1.000 x g für 5 min bei 4 ° C, Kerne und Zelltrümmer zu erhalten.</li>
	</ol></li> <li>Den Überstand zu entfernen und das Pellet in frischen 5 mL Hypotonische Puffer aufzuwirbeln. Filtern der lysate durch ein Sieb 70 µm Zelle in ein 50 mL-Tube. Filtern Sie erneut das Filtrat durch ein Sieb 40 µm Zelle.</li>
	<li>Transfer das Filtrat eine 15 mL Tube und Zentrifuge bei 1.000 x g für 5 Minuten bei 4 ° C, das nukleare Pellet zu erhalten. 
	Hinweis: In diesem Stadium die Kerne können sofort beschallt oder fangen, als eine trockene Pellets in flüssigem Stickstoff eingefroren und bei-80 gelagert ° C.</li>
</ol> 5. Ultraschall <ol><li>das Volumen des nuklearen Geschosses (rund 50 µL für Maschinen- und Dounce Homogenisierung) zu bewerten und in Beschallung Puffer bis zu 3 bis 4 Mal von nuklearen Packvolumen Aufschwemmen und beschallen für 10-15 min bei 4 ° C mit einem Sonikator . 
	Hinweis: Das Chromatin kann sofort analysiert, wie unten (6) beschrieben oder eingefroren und bei-80 ° c</li>
</ol> 6. Beurteilung der Chromatin-Fragmentgröße nach Beschallung Hinweis: siehe Tabelle 1 für alle Kompositionen Puffern.
<ol><li>, De-Crosslink, 30 µL des Chromatins im Reagenzglas 1,5 mL und fügen 20 µL 5 M NaCl. Füllen Sie das Volume 500 µL und über Nacht bei 65 ° C inkubieren.
	<ol><li>Am nächsten Tag, führen Sie eine Behandlung mit 1 µL Proteinase K (20 mg/mL Brühe), 10 µL 2 M Tris pH 6,8 und 10 µL 0,5 M EDTA für 1 h bei 42 ° c führen eine Phenol-Chloroform/Chloroform Extraktion und überstürzen sich die DNA mit 1 Volumen von Natrium-acetat e (3 M) und 2 Bände von 100 % Ethanol für 1 h bei-80 ° C oder über Nacht bei-20 ° c</li>
	</ol></li> <li>Pellet-DNA durch Zentrifugation bei 13.500 x g für 15 min. Dekantieren des Überstandes, waschen das Pellet gründlich mit kaltem 70 % igem Ethanol und Zentrifuge wieder für 5 min. Dekantieren des Überstandes und an der Luft trocknen die Pellet.</li>
	<li>Aufschwemmen der Pellets in das ursprüngliche Volume (30 µL) von TE-Puffer. Die DNA-Konzentration in einem Spektralphotometer durch Absorption bei 260 nm/280 nm zu messen. Pipette 500-1.000 ng DNA zusammen mit einem DNA-Größe Leiter auf getrennten Bahnen eine 1,5 % Agarose-Gel und Elektrophorese zur Beurteilung der Fragmentgröße des Chromatins führen. 
	Hinweis: Fragmentgröße sollte zwischen 200-500 Basenpaaren, und es darf keine nachweisbaren hochmolekularen Fragmente entspricht, nicht oder schlecht-fragmentierten DNA. Bei Bedarf kann die Chromatin-Lösung (Schritt 5) wieder für eine längere Zeit beschallt und analysiert dann erneut wie oben beschrieben, bis die optimale Größe erreicht ist.</li>
</ol> 7. Chromatin Immunopräzipitation (ChIP) Hinweis: siehe Tabelle 1 für alle Kompositionen Puffern.
<ol><li>Block Eiweiß G Sepharose Perlen durch Aliquotierung 1 mL 50 % Gülle in Ethanol. Pellets die Perlen durch Zentrifugation bei 400 X g für 1 min in einem Benchtop Zentrifugieren und waschen mit 1 mL TE-Puffer-Zentrifuge bei 400 X g und wiederholen Sie die Wash
	<ol><li>, Nachdem der zweite Zentrifugation, neu in 1 mL der ChIP Verdünnungspuffer mit 25 µL der BSA (Lager 20 mg/mL) und 20 µL tRNA (Lager 10 mg/mL Hefe). Blockieren Sie die Perlen für mindestens 2 Stunden vor der Verwendung durch Rotation (40-60 u/min) bei 4 ° c</li>
		<li>Verdünnen 50 µg des Chromatins (Schritt 5) 8 bis 10 Mal mit ChIP Verdünnungspuffer. Fügen Sie 50 µL blockierte Wulst Gülle und inkubieren 2 h drehen (40-60 u/min) bei 4 ° C. Zentrifuge bei 400 X g bei 4 ° C zu erhalten bereits geräumten Chromatin und überträgt es auf ein frisches Tubus.</li>
	</ol></li> <li>Für Immunopräzipitation, fügen Sie die entsprechende Menge an primären Antikörper (z. B. 1-2 µg pro 10 µg von Chromatin) und inkubieren Sie über Nacht mit Rotation bei 4 ° c 
	Hinweis: Die optimale Menge des Antikörpers kann vom Lieferanten empfohlen werden oder kann durch ChIP-qPCR mit unterschiedlichen Mengen von Antikörpern durchführen, bis optimale Bereicherung beobachtet wird empirisch ermittelt werden. Auch Eiweiß G Sepharose durch Protein A Sepharose je nach Subtyp des Antikörpers verwendet ersetzt werden kann.</li>
	<li>Fügen Sie die gesperrten Perlen am nächsten Tag und inkubieren Sie für 1 h mit Drehung (40-60 u/min) bei 4 ° C. Zentrifuge bei 400 X g für 20-30 s pellet-die Perlen, überstand zu entfernen, und starten den ChIP wäscht.</li>
	<li>ChIP Wäschen: führen Sie die folgenden Waschungen mit dem Puffer: einmal mit niedrig-Salz-Puffer, und dann zweimal mit hohen Salz Puffer, zweimal mit LiCl Puffer, und schließlich zweimal mit TE-Puffer.
	<ol><li>Jedem Waschgang für 10 min mit Drehung (40-60 u/min) bei 4 ° C durchführen und die Perlen zwischen jedem Waschgang durch Zentrifugation bei 400 X g für 20-30 s in einer Zentrifuge Benchtop-pellet.</li>
	</ol></li> <li>Für Elution, entfernen die letzten Wäsche und die Perlen in 250 µL Elution Buffer (frisch zubereitet) für 15 min bei Raumtemperatur unter Schütteln Aufschwemmen.</li>
	<li>Zentrifuge bei 400 x g und sammeln das Eluat in einem frischen Rohr. Führen Sie diesen Schritt zweimal und dann de-Crosslink das Eluat über Nacht mit 20 µL NaCl 5 M und 1 µL RNase A (10 mg/mL), Proteinase K und Phenol-Chloroform/Chloroform Extr behandelnhandeln wie in Schritt 6.</li>
	<li>In 50 µL TE-Puffer Aufschwemmen und Aliquote für ChIP-qPCR nach Standardprotokollen 15 um die Qualität des Chips.</li>
</ol>

<ol>
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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+Transcription+Regulatory+Networks+with+ChIP-seq+and+RNA-seq.">Mapping Transcription Regulatory Networks with ChIP-seq and RNA-seq.</a> Adv Exp Med Biol. 883, 119-134 (2015).</li><li>Alpern, 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=TAF4,+a+subunit+of+transcription+factor+II+D,+directs+promoter+occupancy+of+nuclear+receptor+HNF4A+during+post-natal+hepatocyte+differentiation.">TAF4, a subunit of transcription factor II D, directs promoter occupancy of nuclear receptor HNF4A during post-natal hepatocyte differentiation.</a> Elife. 3, e03613 (2014).</li><li>Martianov, I., 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=Cell-specific+occupancy+of+an+extended+repertoire+of+CREM+and+CREB+binding+loci+in+male+germ+cells.">Cell-specific occupancy of an extended repertoire of CREM and CREB binding loci in male germ cells.</a> BMC Genomics. 11, 530 (2010).</li><li>Achour, M., 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=Neuronal+identity+genes+regulated+by+super-enhancers+are+preferentially+down-regulated+in+the+striatum+of+Huntington's+disease+mice.">Neuronal identity genes regulated by super-enhancers are preferentially down-regulated in the striatum of Huntington's disease mice.</a> Hum Mol Genet. 24 (12), 3481-3496 (2015).</li><li>Joshi, 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=TEAD+transcription+factors+are+required+for+normal+primary+myoblast+differentiation+in+vitro+and+muscle+regeneration+in+vivo.">TEAD transcription factors are required for normal primary myoblast differentiation in vitro and muscle regeneration in vivo.</a> PLoS Genet. 13 (2), e1006600 (2017).</li><li>Ohkawa, Y., Mallappa, C., Dacwag-Vallaster, C. S., Imbalzano, A. N., DiMario, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Myogenesis: Methods and protocols. 798, 517-531 (2012).</li><li>Dimauro, I., Pearson, T., Caporossi, D., Jackson, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+protocol+for+the+subcellular+fractionation+of+skeletal+muscle+cells+and+tissue.">A simple protocol for the subcellular fractionation of skeletal muscle cells and tissue.</a> BMC Res Notes. 5, 513 (2012).</li><li>Thomas, J. 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=PAK1+and+CtBP1+Regulate+the+Coupling+of+Neuronal+Activity+to+Muscle+Chromatin+and+Gene+Expression.">PAK1 and CtBP1 Regulate the Coupling of Neuronal Activity to Muscle Chromatin and Gene Expression.</a> Mol Cell Biol. 35 (24), 4110-4120 (2015).</li><li>Kuo, T., 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+analysis+of+glucocorticoid+receptor-binding+sites+in+myotubes+identifies+gene+networks+modulating+insulin+signaling.">Genome-wide analysis of glucocorticoid receptor-binding sites in myotubes identifies gene networks modulating insulin signaling.</a> Proc Natl Acad Sci U S A. 109 (28), 11160-11165 (2012).</li><li>Whyte, W. A., 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=Master+transcription+factors+and+mediator+establish+super-enhancers+at+key+cell+identity+genes.">Master transcription factors and mediator establish super-enhancers at key cell identity genes.</a> Cell. 153 (2), 307-319 (2013).</li><li>Benhaddou, A., Keime, C., Ye, T., Morlon, A., Michel, I., Jost, B., Mengus, G., Davidson, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcription+factor+TEAD4+regulates+expression+of+myogenin+and+the+unfolded+protein+response+genes+during+C2C12+cell+differentiation.">Transcription factor TEAD4 regulates expression of myogenin and the unfolded protein response genes during C2C12 cell differentiation.</a> Cell Death and Differentiation. 19 (2), 220-231 (2011).</li><li>Cowling, B. 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=Reducing+dynamin+2+expression+rescues+X-linked+centronuclear+myopathy.">Reducing dynamin 2 expression rescues X-linked centronuclear myopathy.</a> J Clin Invest. 124 (3), 1350-1363 (2014).</li><li>. ChIP Analysis Available from: <a target="_blank" href="https://www.thermofisher.com/fr/fr/home/life-science/epigenetics-noncoding-rna-research/chromatin-remodeling/chromatin-immunoprecipitation-chip/chip-analysis.html">https://www.thermofisher.com/fr/fr/home/life-science/epigenetics-noncoding-rna-research/chromatin-remodeling/chromatin-immunoprecipitation-chip/chip-analysis.html</a> (2017)</li></ol>

Die Autoren erklären, dass sie keine finanziellen Interessenkonflikte.

Hier beschreiben wir eine neuartige Protokoll für die Zubereitung von Chromatin von Erwachsenen Maus Skelettmuskeln und zeigen, dass das Chromatin für ChIP Experimente geeignet ist, die Transkription Faktor Bindung und kovalente Histon-Modifikationen in den Muskelfaser-Kernen zu erkennen. Dieses Protokoll beinhaltet mehrere kritische Schritte. Die erste ist Gewebe Störungen, die durch Dounce oder durch mechanische Scherung erfolgen kann. Mechanische Scherung ist schneller und mehr reproduzierbar, und ist daher die Met...

Chromatin Immunopräzipitation (ChIP) mit quantitativen Polymerase-Kettenreaktion (qPCR) gekoppelt und Hochdurchsatz-Sequenzierung (ChIP-Seq) geworden, die Methoden der Wahl Transkription zu studieren und epigenetischen Regulation der Genexpression in verschiedenen Geweben und Zelltypen1. Diese Technik erlaubt es genomweiten Profilierung der kovalente Chromatin Änderungen, Histon Variante Belegung und Transkription Faktor Bindung2,3.
ChIP von kultivierten Zellen gut etabliert ist, bleibt ChIP von Säugetier-Gewebe schwieriger. Die Vorbereitung von Chromatin für ChIP umfasst mehrere wichtige Schritte, die für jede Art von Gewebe und Zellen optimiert werden müssen. Chromatin kann aus der zellulären Lysates kultivierten Zellen oder folgende Reinigung des ihre Kerne hergestellt werden. Im Falle von Säugetier-Gewebe sind effiziente lyse, Formaldehyd Fixierung und Reinigung der Kerne kritisch, um optimale Erholung des Chromatins zu gewährleisten. Darüber hinaus hat die Wahl, ob die Kerne vor oder nach ihrer Reinigung beheben experimentell zu ermitteln. Trotz dieser Hürden hat ChIP aus dem Gewebe wie Leber, Hoden oder Gehirn4,5,6erfolgreich durchgeführt. Im Falle der Skelettmuskulatur ist Störung der physisch resistent Gewebe, die einen hohen Gehalt an Strukturproteinen und Isolation von seinen Kernen Exponate besonders anspruchsvoll. Angesichts dieser Besonderheit, geben Protokolle, optimiert für andere Gewebe nicht zufriedenstellende Ergebnisse für skelettartigen Muskeln.
Hier beschreiben wir ein Protokoll zur ChIP-Grade Chromatin von Maus Skelettmuskelgewebe zu isolieren, die physisch stören das Gewebe, Formaldehyd Fixierung und die Isolation der Kerne und Beschallung beinhaltet. Die Effizienz dieser Methode Chromatin geeignet für ChIP aus diesem Gewebe vorzubereiten zeigte sich durch ChIP-qPCR und ChIP-Seq für verschiedene Transkriptionsfaktoren, RNA-Polymerase II und kovalente Histon-Modifikationen-7.
Diese neue Technik ist wesentlich schneller als ein zuvor gemeldeten Protokoll8 bestehend aus lange Kollagenase Verdauung Schritte während dieser Zeit, Änderungen bei genomischen Lokalisierung von Transkriptionsfaktoren und Veränderungen in der Genexpression erfolgen können. Die Schnelligkeit, mit der die Kerne isoliert und fixiert sind, macht die hier beschriebene Methode besonders attraktiv, Chromatin vorzubereiten, die mehr treu den nativen genomische Belegung Zustand erfasst. Außerdem, obwohl andere Methoden für Chromatin Isolierung oder Protein-Extraktion aus Muskelgewebe haben beschrieben8,9,10 und verwendet wurden für ChIP-qPCR Experimente auf ausgewählte Gene, kein ChIP-seq Daten wurde über sie berichtet. Die Methode hier berichtet eignet sich für Transkription Faktor und Histon Modifikation ChIP-Seq, und daher sollte auch für 3 / 4C oder HiC Chromatin Konformation Erfassung Anwendungen geeignet.

<table>
	<tbody>
		<tr>
			<td>T 25 digital ULTRA-TURRAX</td>
			<td>T 18 ULTRA-TURRAX</td>
			<td>0009022800</td>
			<td></td>
		</tr>
		<tr>
			<td>PMSF</td>
			<td>SIGMA-ALDRICH CHIMIE SARL</td>
			<td>P7626-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease Inhibitor Cocktail-EDTA Free</td>
			<td>Roche Diagnostics</td>
			<td>11873580001</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein G Sepharose beads</td>
			<td>SIGMA-ALDRICH CHIMIE</td>
			<td>P-3296</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>SIGMA-ALDRICH CHIMIE</td>
			<td>P-2308</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Strainers 70um</td>
			<td>Corning BV</td>
			<td>431751</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Strainers 40um</td>
			<td>Corning BV</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde EM grade</td>
			<td>Euromedex</td>
			<td>15710-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Rnase A</td>
			<td>Fischer Scientific</td>
			<td>12091039</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol:Chloroform:Isoamyl Alcohol 25:24:1</td>
			<td>SIGMA-ALDRICH CHIMIE</td>
			<td>P3803</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>SIGMA-ALDRICH CHIMIE</td>
			<td>B4287-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>yeast tRNA</td>
			<td>SIGMA-ALDRICH CHIMIE</td>
			<td>R5636</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycogen Blue</td>
			<td>AMIBION</td>
			<td>AM9516</td>
			<td></td>
		</tr>
		<tr>
			<td>Pol II ChIP Antibody</td>
			<td>Santa Cruz</td>
			<td>SC-9001</td>
			<td></td>
		</tr>
		<tr>
			<td>H3K27ac ChIP Antibody</td>
			<td>Active Motif</td>
			<td>39133</td>
			<td></td>
		</tr>
		<tr>
			<td>Tead4 ChIP Antibody</td>
			<td>Aviva Systems Biology</td>
			<td>(ARP38276_P050)</td>
			<td></td>
		</tr>
		<tr>
			<td>IGEPAL</td>
			<td>SIGMA-ALDRICH CHIMIE</td>
			<td>I-3021</td>
			<td></td>
		</tr>
		<tr>
			<td>E220 Focused Ultrasonicator</td>
			<td>Covaris E220</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Additional items</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Loose Dounce</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml and 50 ml falcon tubes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>14 ml round bottom tubes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 ml and 2 ml Eppendorf tubes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#956;m and 40 &#956;m cell strainers (Corning)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

improved protocol for chromatin immunoprecipitation from mouse skeletal muscle

Wir beschreiben eine effiziente und reproduzierbare Protokoll für die Vorbereitung von Chromatin von Erwachsenen Maus Skelettmuskulatur, eine physisch resistent Gewebe mit einem hohen Gehalt von Strukturproteinen. Sezierten Gliedmaßen Muskeln von Erwachsenen Mäusen sind physisch durch mechanische Homogenisierung oder eine Kombination von hacken und Douncing in einem hypotonischen Puffer vor Formaldehyd Fixierung der Zelle lysate gestört. Die feste Kerne werden gereinigt, durch weitere Zyklen von mechanischer Homogenisierung oder Douncing und sequentielle Filtrationen, Zelle Ablagerungen zu entfernen. Die gereinigte Kerne können sofort oder zu einem späteren Zeitpunkt nach dem Einfrieren beschallt werden. Das Chromatin kann effizient beschallt und ist geeignet für Chromatin Immunopräzipitation Experimente, wie durch die Profile für Transkriptionsfaktoren, RNA-Polymerase II und kovalente Histon Änderungen erhalten. Die verbindliche Ereignisse mit Chromatin vorbereitet durch dieses Protokoll nachgewiesen sind überwiegend in die Muskelfaser Kerne trotz der Anwesenheit von Chromatin von anderen Faser-assoziierten Satelliten- und Endothelzellen stattfindet. Dieses Protokoll ist daher angepasst, Genregulation in der Skelettmuskulatur erwachsener Mäuse zu studieren.

Um die Kerne zu isolieren, führten wir mechanische Homogenisierung des Muskelgewebes seziert und Hackfleisch für entweder 15 oder 45 s, bei 18.000 und 22.000 u/min (siehe Tabelle der Materialien). Unter allen Bedingungen Kerne aus den Trümmern Gewebe getrennt werden könnte, aber die Ausbeute war optimal mit der niedrigeren Drehzahl (Abbildung 1A-B). Kerne könnten auch durch Douncing für 3-5 min (Abb...

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Improved Protocol for Chromatin Immunoprecipitation from Mouse Skeletal Muscle

Verbesserte Protokoll für Chromatin Immunopräzipitation aus Maus skelettartiger Muskel

Eine neuartige Protokoll für die Vorbereitung von Chromatin von Erwachsenen Maus Skelettmuskulatur zur Erforschung der Genregulation in Muskelfasern von Chromatin Immunopräzipitation angepasst präsentiert.

We describe an efficient and reproducible protocol for the preparation of chromatin from adult mouse skeletal muscle, a physically resistant tissue with a ...

improved-protocol-for-chromatin-immunoprecipitation-from-mouse

Research

JoVE Journal

Biochemistry

8.4K Aufrufe.  Institut de Génétique et de Biologie Moléculaire et Cellulaire. Eine neuartige Protokoll für die Vorbereitung von Chromatin von Erwachsenen Maus Skelettmuskulatur zur Erforschung der Genregulation in Muskelfasern von Chromatin Immunopräzipitation angepasst präsentiert.

Serie: Improved Protocol for Chromatin Immunoprecipitation from Mouse Skeletal Muscle

Optimized Protocol for the Extraction of Proteins from the Human Mitral Valve

Analysis of the cellular proteome can help to elucidate the molecular mechanisms underlying diseases due to the development of technologies that permit the large-scale identification and quantification of the proteins present in complex biological systems.The knowledge gained from a proteomic approach can potentially lead to a better understanding of the pathogenic mechanisms underlying diseases, allowing for the identification of novel diagnostic and prognostic disease markers, and, hopefully, of therapeutic targets. However, the cardiac mitral valve represents a very challenging sample for proteomic analysis because of the low cellularity in proteoglycan and collagen-enriched extracellular matrix. This makes it challenging to extract proteins for a global proteomic analysis. This work describes a protocol that is compatible with subsequent protein analysis, such as quantitative proteomics and immunoblotting. This can allow for the correlation of data concerning protein expression with data on quantitative mRNA expression and non-quantitative immunohistochemical analysis. Indeed, these approaches, when performed together, will lead to a more comprehensive understanding of the molecular mechanisms underlying diseases, from mRNA to post-translational protein modification. Thus, this method can be relevant to researchers interested in the study of cardiac valve physiopathology.

The protein composition of the human mitral valve is still partially unknown, because its analysis is complicated by low cellularity and therefore by low protein biosynthesis. This work provides a protocol to efficiently extract protein for the analysis of the mitral valve proteome.

Optimiertes Protokoll zur Extraktion von Proteinen aus dem menschlichen Mitralventil

Analysis of the cellular proteome can help to elucidate the molecular mechanisms underlying diseases due to the development of technologies that permit ...

Forschung

Biochemie

A Filtration-based Method of Preparing High-quality Nuclei from Cross-linked Skeletal Muscle for Chromatin Immunoprecipitation

Chromatin immunoprecipitation (ChIP) is a powerful method to determine protein binding to chromatin DNA. Fiber-rich skeletal muscle, however, has been a challenge for ChIP due to technical difficulty in isolation of high-quality nuclei with minimal contamination of myofibrils. Previous protocols have attempted to purify nuclei before cross-linking, which incurs the risk of altered DNA-protein interaction during the prolonged nuclei preparation process. In the current protocol, we first cross-linked the skeletal muscle tissue collected from mice, and the tissues were minced and sonicated. Since we found that ultracentrifugation was not able to separate nuclei from myofibrils using cross-linked muscle tissue, we devised a sequential filtration procedure to obtain high-quality nuclei devoid of significant myofibril contamination. We subsequently prepared chromatin by using an ultrasonicator, and ChIP assays with anti-BMAL1 antibody revealed robust circadian binding pattern of BMAL1 to target gene promoters. This filtration protocol constitutes an easily applicable method to isolate high-quality nuclei from cross-linked skeletal muscle tissue, allowing consistent sample processing for circadian and other time-sensitive studies. In combination with next-generation sequencing (NGS), our method can be deployed for various mechanistic and genomic studies focusing on skeletal muscle function.

We present a filtration-based protocol to isolate high-quality nuclei from cross-linked mouse skeletal muscle wherein we removed the need for ultracentrifugation, making it easily applicable. We show that chromatin prepared from the nuclei is suitable for chromatin immunoprecipitation and likely chromatin immunoprecipitation sequencing studies.

Eine Filtrations-basierte Methode zur Herstellung von hochwertigen Nuklei aus vernetzten Skelettmuskeln für Chromatin-Immunpräzipitation

Chromatin immunoprecipitation (ChIP) is a powerful method to determine protein binding to chromatin DNA. Fiber-rich skeletal muscle, however, has been a ...

A Western Blotting Protocol for Small Numbers of Hematopoietic Stem Cells

Hematopoietic stem cells (HSCs) are rare cells, with the mouse bone marrow containing only ~25,000 phenotypic long term repopulating HSCs. A Western blotting protocol was optimized and suitable for the analysis of small numbers of HSCs (500 - 15,000 cells). Phenotypic HSCs were purified, accurately counted, and directly lysed in Laemmli sample buffer. Lysates containing equal numbers of cells were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the blot was prepared and processed following standard Western blotting protocols. Using this protocol, 2,000 - 5,000 HSCs can be routinely analyzed, and in some cases data can be obtained from as few as 500 cells, compared to the 20,000 to 40,000 cells reported in most publications. This protocol should be generally applicable to other hematopoietic cells, and enables the routine analysis of small numbers of cells using standard laboratory procedures.

A standard Western blotting protocol was optimized for analyzing as few as 500 hematopoietic stem or progenitor cells. Optimization involves careful handling of the cell sample, limiting transfers between tubes, and directly lysing the cells in Laemmli sample buffer.

Ein Western-Blot-Protokoll für eine kleine Zahl von Hämatopoetischen Stammzellen

Hematopoietic stem cells (HSCs) are rare cells, with the mouse bone marrow containing only ~25,000 phenotypic long term repopulating HSCs. A Western ...

Utilizing a Comprehensive Immunoprecipitation Enrichment System to Identify an Endogenous Post-translational Modification Profile for Target Proteins

It is now well-appreciated that post-translational modifications (PTMs) play an integral role in regulating a protein&#39;s structure and function, which may be essential for a given protein&#39;s role both physiologically and pathologically. Enrichment of PTMs is often necessary when investigating the PTM status of a target protein, because PTMs are often transient and relatively low in abundance. Many pitfalls are encountered when enriching for a PTM of a target protein, such as buffer incompatibility, the target protein antibody is not IP-compatible, loss of PTM signal, and others. The degree of difficulty is magnified when investigating multiple PTMs like acetylation, ubiquitination, SUMOylation 2/3, and tyrosine phosphorylation for a given target protein. Studying a combination of these PTMs may be necessary, as crosstalk between PTMs is prevalent and critical for protein regulation. Often, these PTMs are studied in different lysis buffers and with unique inhibitor compositions. To simplify the process, a unique denaturing lysis system was developed that effectively isolates and preserves these four PTMs; thus, enabling investigation of potential crosstalk in a single lysis system. A unique filter system was engineered to remove contaminating genomic DNA from the lysate, which is a problematic by-product of denaturing buffers. Robust affinity matrices targeting each of the four PTMs were developed in concert with the buffer system to maximize the enrichment and detection of the endogenous states of these four PTMs. This comprehensive PTM detection toolset streamlines the process of obtaining critical information about whether a protein is modified by one or more of these PTMs.

Investigating multiple, endogenous post-translational modifications for a target protein can be extremely challenging. Techniques described here utilize an optimized lysis buffer and filter system developed with specific PTM-targeting, affinity matrices to detect the acetylation, ubiquitination, SUMOylation 2/3, and tyrosine phosphorylation modifications for a target protein using a single, streamlined system.

Nutzung eines umfassenden Immunopräzipitation Bereicherung System um eine endogene Post-translationale Modifikation Profil für Zielproteine zu identifizieren

It is now well-appreciated that post-translational modifications (PTMs) play an integral role in regulating a protein&#39;s structure and function, which ...

Measuring and Interpreting Oxygen Consumption Rates in Whole Fly Head Segments

Regulated metabolic activity is essential for the normal functioning of living cells. Indeed, altered metabolic activity is causally linked with the progression of cancer, diabetes, neurodegeneration, and aging to name a few. For instance, changes in mitochondrial activity, the cell&#39;s metabolic powerhouse, have been characterized in many such diseases. Generally, the oxygen consumption rates of mitochondria were considered a reliable readout of mitochondrial activity and measurements in some of these studies were based on isolated mitochondria or cells. However, such conditions may not represent the complexity of a whole tissue. Recently, we have developed a novel method that enables the dynamic measurement of oxygen consumption rates from whole isolated fly heads. By utilizing this method, we have recorded lower oxygen consumption rates of the whole head segment in young versus aged flies. Secondly, we have discovered that lysine deacetylase inhibitors rapidly alter the oxygen consumption in the whole head. Our novel technique may therefore aid in uncovering new properties of various drugs, which may impact metabolic rates. Furthermore, our method may give a better understanding of metabolic behavior in an experimental setup that more closely resembles physiological states.

Measuring alterations in metabolic rates is central to understanding the progression of various diseases and aging. Here, we present a novel technique to measure whole head oxygen consumption that more closely resembles the physiological state and may aid in revealing novel drugs that modify mitochondrial activity.

Messung und Interpretation von Sauerstoff-Verbrauch im ganzen fliegen Kopf Segmente

Regulated metabolic activity is essential for the normal functioning of living cells. Indeed, altered metabolic activity is causally linked with the ...

Quantification of Protein Interaction Network Dynamics using Multiplexed Co-Immunoprecipitation

Dynamic protein-protein interactions control cellular behavior, from motility to DNA replication to signal transduction. However, monitoring dynamic interactions among multiple proteins in a protein interaction network is technically difficult. Here, we present a protocol for Quantitative Multiplex Immunoprecipitation (QMI), which allows quantitative assessment of fold changes in protein interactions based on relative fluorescence measurements of Proteins in Shared Complexes detected by Exposed Surface epitopes (PiSCES). In QMI, protein complexes from cell lysates are immunoprecipitated onto microspheres, and then probed with a labeled antibody for a different protein in order to quantify the abundance of PiSCES. Immunoprecipitation antibodies are conjugated to different MagBead spectral regions, which allows a flow cytometer to differentiate multiple parallel immunoprecipitations and simultaneously quantify the amount of probe antibody associated with each. QMI does not require genetic tagging and can be performed using minimal biomaterial compared to other immunoprecipitation methods. QMI can be adapted for any defined group of interacting proteins, and has thus far been used to characterize signaling networks in T cells and neuronal glutamate synapses. Results have led to new hypothesis generation with potential diagnostic and therapeutic applications. This protocol includes instructions to perform QMI, from the initial antibody panel selection through to running assays and analyzing data. The initial assembly of a QMI assay involves screening antibodies to generate a panel, and empirically determining an appropriate lysis buffer. The subsequent reagent preparation includes covalently coupling immunoprecipitation antibodies to MagBeads, and biotinylating probe antibodies so they can be labeled by a streptavidin-conjugated fluorophore. To run the assay, lysate is mixed with MagBeads overnight, and then beads are divided and incubated with different probe antibodies, and then a fluorophore label, and read by flow cytometry. Two statistical tests are performed to identify PiSCES that differ significantly between experimental conditions, and results are visualized using heatmaps or node-edge diagrams.

Quantitative Multiplex Immunoprecipitation (QMI) uses flow cytometry for sensitive detection of differences in the abundance of targeted protein-protein interactions between two samples. QMI can be performed using a small amount of biomaterial, does not require genetically engineered tags, and can be adapted for any previously defined protein interaction network.

Quantifizierung der Protein-Interaktionsnetzwerkdynamik mit Multiplex-Co-Immunopräzipitation

Dynamic protein-protein interactions control cellular behavior, from motility to DNA replication to signal transduction. However, monitoring dynamic ...

Label-Free Immunoprecipitation Mass Spectrometry Workflow for Large-scale Nuclear Interactome Profiling

Immunoaffinity purification mass spectrometry (IP-MS) has emerged as a robust quantitative method of identifying protein-protein interactions. This publication presents a complete interaction proteomics workflow designed for identifying low abundance protein-protein interactions from the nucleus that could also be applied to other subcellular compartments. This workflow includes subcellular fractionation, immunoprecipitation, sample preparation, offline cleanup, single-shot label-free mass spectrometry, and downstream computational analysis and data visualization. Our protocol is optimized for detecting compartmentalized, low abundance interactions that are difficult to identify from whole cell lysates (e.g., transcription factor interactions in the nucleus) by immunoprecipitation of endogenous proteins from fractionated subcellular compartments. The sample preparation pipeline outlined here provides detailed instructions for the preparation of HeLa cell nuclear extract, immunoaffinity purification of endogenous bait protein, and quantitative mass spectrometry analysis. We also discuss methodological considerations for performing large-scale immunoprecipitation in mass spectrometry-based interaction profiling experiments and provide guidelines for evaluating data quality to distinguish true positive protein interactions from nonspecific interactions. This approach is demonstrated here by investigating the nuclear interactome of the CMGC kinase, DYRK1A, a low abundance protein kinase with poorly defined interactions within the nucleus.

Described is a proteomics workflow for identifying protein interaction partners from a nuclear subcellular fraction using immunoaffinity enrichment of a given protein of interest and label-free mass spectrometry. The workflow includes subcellular fractionation, immunoprecipitation, filter aided sample preparation, offline cleanup, mass spectrometry, and a downstream bioinformatics pipeline.

Etikettenfreier Immunpräzipitations-Massenspektrometrie-Workflow für großangelegtes Nuklear-Interactome-Profiling

Immunoaffinity purification mass spectrometry (IP-MS) has emerged as a robust quantitative method of identifying protein-protein interactions. This ...

A Rapidly Incremented Tethered-Swimming Maximal Protocol for Cardiorespiratory Assessment of Swimmers

Incremental exercise testing is the standard means of assessing cardiorespiratory capacity of endurance athletes. While the maximal rate of oxygen consumption is typically used as the criterion measurement in this regard, two metabolic breakpoints that reflect changes in the dynamics of lactate production/consumption as the work rate is increased are perhaps more relevant for endurance athletes from a functional standpoint. Exercise economy, which represents the rate of oxygen consumption relative to performance of submaximal work, is also an important parameter to measure for endurance-athlete assessment. Ramp incremental tests comprising a gradual but rapid increase in work rate until the limit of exercise tolerance is reached are useful for determining these parameters. This type of test is typically performed on a cycle ergometer or treadmill because there is a need for precision with respect to work-rate incrementation. However, athletes should be tested while performing the mode of exercise required for their sport. Consequently, swimmers are typically assessed during free-swimming incremental tests where such precision is difficult to achieve. We have recently suggested that stationary swimming against a load that is progressively increased (incremental tethered swimming) can serve as a &#34;swim ergometer&#34; by allowing sufficient precision to accommodate a gradual but rapid loading pattern that reveals the aforementioned metabolic breakpoints and exercise economy. However, the degree to which the peak rate of oxygen consumption achieved during such a protocol approximates the maximal rate that is measured during free swimming remains to be determined. In the present article, we explain how this rapidly incremented tethered-swimming protocol can be employed to assess the cardiorespiratory capacity of a swimmer. Specifically, we explain how assessment of a short-distance competitive swimmer using this protocol revealed that his rate of oxygen uptake was 30.3 and 34.8 mL&#8729;min-1&#8729;kg-1BM at his gas-exchange threshold and respiratory compensation point, respectively.

As opposed to measurement during free swimming, which presents inherent challenges and limitations, determination of important parameters of cardiorespiratory function for swimmers can be made using a more feasible and easier to administer tethered-swimming rapidly incremented protocol with gas exchange and ventilatory data collection.

Ein schnell inkrementiertes Tethered-Swimming-Maximalprotokoll zur kardiorspiratorischen Beurteilung von Schwimmern

Incremental exercise testing is the standard means of assessing cardiorespiratory capacity of endurance athletes. While the maximal rate of oxygen ...

RIBO-seq in Bacteria: a Sample Collection and Library Preparation Protocol for NGS Sequencing

The ribosome profiling technique (RIBO-seq) is currently the most effective tool for studying the process of protein synthesis in vivo. The advantage of this method, in comparison to other approaches, is its ability to monitor translation by precisely mapping the position and number of ribosomes on a mRNA transcript.
In this article, we describe the consecutive stages of sample collection and preparation for RIBO-seq method in bacteria, highlighting the details relevant to the planning and execution of the experiment.
Since the RIBO-seq relies on intact ribosomes and related mRNAs, the key step is rapid inhibition of translation and adequate disintegration of cells. Thus, we suggest filtration and flash-freezing in liquid nitrogen for cell harvesting with an optional pretreatment with chloramphenicol to arrest translation in bacteria. For the disintegration, we propose grinding frozen cells with mortar and pestle in the presence of aluminum oxide to mechanically disrupt the cell wall. In this protocol, sucrose cushion or a sucrose gradient ultracentrifugation for monosome purification is not required. Instead, mRNA separation using polyacrylamide gel electrophoresis (PAGE) followed by the ribosomal footprint excision (28-30 nt band) is applied and provides satisfactory results. This largely simplifies the method as well as reduces the time and equipment requirements for the procedure. For library preparation, we recommend using the commercially available small RNA kit for Illumina sequencing from New England Biolabs, following manufacturer&#39;s guidelines with some degree of optimization.
The resulting cDNA libraries present appropriate quantity and quality required for next generation sequencing (NGS). Sequencing of the libraries prepared according to the described protocol results in 2 to 10 mln uniquely mapped reads per sample providing sufficient data for comprehensive bioinformatic analysis. The protocol we present is quick and relatively easy and can be performed with standard laboratory equipment.

Here we describe the stages of sample collection and preparation for RIBO-seq in bacteria. Sequencing of the libraries prepared according to these guidelines results in sufficient data for comprehensive bioinformatic analysis. The protocol we present is simple, uses standard laboratory equipment and takes seven days from lysis to obtaining libraries.

RIBO-seq in Bakterien: ein Probensammlungs- und Bibliotheksvorbereitungsprotokoll für die NGS-Sequenzierung

The ribosome profiling technique (RIBO-seq) is currently the most effective tool for studying the process of protein synthesis in vivo. The advantage of ...

Measurement of Protein Import Capacity of Skeletal Muscle Mitochondria

Mitochondria are key metabolic and regulatory organelles that determine the energy supply as well as the overall health of the cell. In skeletal muscle, mitochondria exist in a series of complex morphologies, ranging from small oval organelles to a broad, reticulum-like network. Understanding how the mitochondrial reticulum expands and develops in response to diverse stimuli such as alterations in energy demand has long been a topic of research. A key aspect of this growth, or biogenesis, is the import of precursor proteins, originally encoded by the nuclear genome, synthesized in the cytosol, and translocated into various mitochondrial sub-compartments. Mitochondria have developed a sophisticated mechanism for this import process, involving many selective inner and outer membrane channels, known as the protein import machinery (PIM). Import into the mitochondrion is dependent on viable membrane potential and the availability of organelle-derived ATP through oxidative phosphorylation. Therefore its measurement can serve as a measure of organelle health. The PIM also exhibits a high level of adaptive plasticity in skeletal muscle that is tightly coupled to the energy status of the cell. For example, exercise training has been shown to increase import capacity, while muscle disuse reduces it, coincident with changes in markers of mitochondrial content. Although protein import is a critical step in the biogenesis and expansion of mitochondria, the process is not widely studied in skeletal muscle. Thus, this paper outlines how to use isolated and fully functional mitochondria from skeletal muscle to measure protein import capacity in order to promote a greater understanding of the methods involved and an appreciation of the importance of the pathway for organelle turnover in exercise, health, and disease.

Mitochondria are key metabolic organelles that exhibit a high level of phenotypic plasticity in skeletal muscle. The import of proteins from the cytosol is a critical pathway for organelle biogenesis, essential for the expansion of the reticulum and the maintenance of mitochondrial function. Therefore, protein import serves as a barometer of cellular health.

Messung der Proteinimportkapazität der Mitochondrien der Skelettmuskulatur

Mitochondria are key metabolic and regulatory organelles that determine the energy supply as well as the overall health of the cell. In skeletal muscle, ...