Name: improved protocol for chromatin immunoprecipitation from mouse skeletal muscle
Uploaded: 2017-11-06T17:00:00
Description: Watch this Scientific Journal Video about Improved Protocol for Chromatin Immunoprecipitation from Mouse Skeletal Muscle at JoVE.com

我们感谢 IGBMC 高吞吐量测序设施的所有工作人员, 即 "法国 Génomique" 财团 (ANR10-INBS-09-08) 的成员和所有 IGBMC 一般事务人员, 特别是 IGBMC 动物设施的工作人员。这项工作得到了来自 CNRS、INSERM、AFM、甲国家组织勒癌症的赠款、法国全国基金在 Investissements 艾文莉标记 ANR-10-IDEX-0002-02、Labex INRT ANR-10-IDEX-0001-02 和ANR-AR2GR-16-CE11-009-01资助者在研究设计、数据收集和分析、决定出版或准备原稿方面没有作用。甲组织的支持下, 美国的 ANR-AR2GR-16-CE11-009-01, 和五, U 由 Ministère de Enseignement 等研究。ID 是一个 ' équipe labellisée ' 的甲国立组织勒癌症。

根据国家动物保育指导方针 (欧洲委员会指令 86/609/中欧和东欧), 按照有关实验室动物的护理和使用的机构准则, 对老鼠进行了保存;法国法令 87-848)。所有程序均由法国国家伦理委员会批准. 
 1. 肌肉组织的分离 <ol> <li> 由颈椎脱位牺牲一成人6至8周大鼠。Sterlise 用70% 乙醇冲洗四肢, 用细点剪刀和镊子解剖后肢肌肉 (腓肠肌、胫骨前、四头肌). 
	注意: 一只老鼠应该产生大约500毫克的组织. </li>
...

<ol>
	<li>Johnson, D. S., Mortazavi, A., Myers, R. M., Wold, B. <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+mapping+of+in+vivo+protein-DNA+interactions.">Genome-wide mapping of in vivo protein-DNA interactions.</a> Science. 316 (5830), 1497-1502 (2007).</li><li>Furey, T. S. <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+beyond:+new+and+improved+methodologies+to+detect+and+characterize+protein-DNA+interactions.">ChIP-seq and beyond: new and improved methodologies to detect and characterize protein-DNA interactions.</a> Nat Rev Genet. 13 (12), 840-852 (2012).</li><li>Wade, J. 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>

在这里, 我们描述了一个新的协议, 从成年小鼠骨骼肌肉制备染色质, 并表明, 这种染色质是适用于芯片实验, 检测转录因子结合和共价键组蛋白修饰肌纤维核。此协议涉及几个关键步骤。第一种是组织破坏, 可以通过 dounce 或机械剪切来进行。机械剪切速度快, 重现性好, 因此是选择的方法。不过, 如果没有合适的仪器, 也可以由 douncing, 在进行固定步骤之前, 建议在显微镜下对裂解效率进行评估。固?...

染色质沉淀 (芯片) 结合定量聚合酶链反应 (qPCR) 和高通量测序 (芯片序列) 已成为选择的方法研究转录和表观遗传调控的基因表达在各种组织和单元格类型1。这项技术允许基因组范围内共价染色质修饰, 蛋白的变体占用, 和转录因子绑定2,3。
虽然从培养细胞中执行芯片是很好的建立, 从哺乳动物组织芯片仍然是更具挑战性。为芯片准备染色质涉及几个关键步骤, 需要优化每一个组织和细胞类型。染色质可以从细胞裂解培养细胞或在其细胞核纯化后制备。在哺乳动物组织的情况下, 有效的裂解, 甲醛固定和纯化的细胞核是至关重要的, 以确保最佳的恢复染色质。此外, 选择是否修复核之前或之后, 他们的纯化必须实验确定。尽管有这些障碍, 芯片已成功地从组织, 如肝脏, 睾丸, 或大脑4,5,6。在骨骼肌肉的情况下, 破坏这种身体上的抗性组织, 它表现出高含量的结构蛋白, 和孤立的细胞核是特别具有挑战性的。鉴于这种特异性, 为其他组织优化的协议不会给骨骼肌肉带来令人满意的结果。
在这里, 我们描述了一个协议, 隔离芯片级染色质从小鼠骨骼肌组织, 包括物理扰乱组织, 甲醛固定, 然后分离细胞核和超声。通过对各种转录因子、RNA 聚合酶 II 和共价组蛋白修饰的芯片 qPCR 和芯片序列进行了研究, 证明了该方法制备适合于该组织芯片的染色质的效率7。
这种新的技术比以前报告的协议8要快得多, 包括长时间胶原酶消化步骤, 在此期间, 转录因子基因组本地化的变化和基因表达的改变可能会发生。原子核被隔绝和固定的快速, 使这里描述的方法特别有吸引力准备染色质更加忠实地捕获本机基因组占有状态。此外, 虽然其他方法的染色质分离或蛋白质提取从肌肉组织已被描述8,9,10 , 并用于芯片 qPCR 实验选定的基因, 没有芯片 seq已报告使用这些数据。所报告的方法适用于转录因子和组蛋白修饰芯片序列, 因此也应适用于 3/4 c 或细胞核染色质构象捕获应用。

<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

我们描述了一个有效的和可复制的协议, 从成年小鼠骨骼肌的染色质制备, 一个具有高含量结构蛋白的身体抗性组织。从成年小鼠身上解剖肢体肌肉被机械化, 或切碎和 douncing 的组合, 在低渗缓冲液中, 在甲醛固定的细胞裂解。固定核通过机械化或 douncing 的进一步循环纯化, 并连续过滤去除细胞碎片。纯化后的核可以立即声或在冷冻后的后期进行。染色质可以有效地声, 适用于染色质沉淀实验, 如为转录因子、RNA 聚合酶 II 和共价键组蛋白修饰所获得的剖面所示。使用本协议所制备的染色质检测到的绑定事件主要是在肌纤维核中发生的, 尽管其他纤维相关的卫星和内皮细胞存在染色质。因此, 本议定书适用于研究成年小鼠骨骼肌的基因调控。

为了分离细胞核, 我们在15或四十五年代, 在1.8万和 2.2万 rpm 进行了解剖和碎肌组织的机械均质化 (参见材料表)。在所有条件下, 细胞核可以与组织的碎片分离, 但产量是最好的使用较低的速度 (图 1A-b)。原子核也可以由 douncing 为 3-5 min (图 1A, 和数据没有显示), 但机械均匀化是选择的方法, 因为最理想的产...

Watch this Scientific Journal Video about Improved Protocol for Chromatin Immunoprecipitation from Mouse Skeletal Muscle at JoVE.com

Improved Protocol for Chromatin Immunoprecipitation from Mouse Skeletal Muscle

改进的小鼠骨骼肌染色质沉淀的协议

本文介绍了一种新的制备成年小鼠骨骼肌染色质的方法, 该方法适合于用染色质沉淀研究肌纤维的基因调控。

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

Research

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