Name: a filtration-based method of preparing high-quality nuclei from cross-linked skeletal muscle for chromatin immunoprecipitation
Uploaded: 2017-07-06T15:00:00
Description: Watch this Scientific Journal Video about A Filtration-based Method of Preparing High-quality Nuclei from Cross-linked Skeletal Muscle for Chromatin Immunoprecipitation at JoVE.com

我们感谢Karyn Esser，Nobuya小池和Noheon Park提供有用的建议。这项工作部分由NIH / NIGMS（R01GM114424）向S.-HY和Robert A.Welch基金会（AU-1731）和NIH / NIA（R01AG045828）向ZC提供支持

动物护理在机构动物护理和使用委员会（IACUC）指南下进行，并且程序是根据得克萨斯州休斯敦大学健康科学中心批准的动物方案进行的。 从交联骨骼肌中分离核<ol><li>称重和切碎后肢骨骼肌，从大约20周龄的C57BL / 6雄性小鼠分离，在冰冷的磷酸盐缓冲盐水（PBS）中，如先前详细描述的22所述 。 注意：我们通常从一只小鼠获得1.0 - 1.5 g骨骼肌。 <ol><li>?...

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在这里，我们描述了一种使用交联骨骼肌组织分离高质量细胞核的可靠方法。进行顺序过滤以有效地将细胞核与碎片分离，来自碟形换能器的超声波能量剪切染色质进行ChIP分析。结果显示BMAL1与靶启动子的昼夜节律时间特异性结合。 当交联发生时，ChIP可用于捕获基因组DNA上的实时蛋白质占用。为了利用这种潜力，我们旨在开发一种方法，允许在组织解剖时交叉骨骼肌，并简?...

骨骼肌在生理和行为中起重要作用。多核肌纤维由肌原纤维组成，其中肌动蛋白和肌球蛋白形成称为sarcomeres的功能单位以产生收缩力。骨骼肌也是身体中最大的代谢器官，占餐后葡萄糖摄入量的80％以上，调节胰岛素反应和代谢体内平衡1,2 。肌肉生理和代谢受到昼夜节律钟的限制，内在的生物计时器3,4,5,6。例如， Bmal1的骨骼肌特异性缺失，核心昼夜节律钟成分之一，导致骨骼肌中胰岛素抵抗和葡萄糖摄取减少，发现动物发生2型糖尿病7 。一世此外，骨骼肌也越来越被认为是内分泌器官8 ，分泌肌动蛋白以调节全身代谢和生理学。机械研究需要完全了解骨骼肌中的这些调节功能。 ChIP是描述DNA结合蛋白启动子募集的有力方法。最初开发了ChIP以鉴定染色质DNA 9,10上的核小体组织。据报道，使用甲醛，硫酸二甲酯或紫外线照射（UV） 11,12来交联蛋白质和染色质DNA的各种方法。甲醛交联是最常用的，保存染色质结构和DNA-蛋白相互作用9,13,14 。交联色谱通过超声处理切片并用针对感兴趣的特定DNA结合蛋白15,16的抗体进行免疫沉淀。近年来，已经开发了ChIP-sequencing（ChIP-seq），一种将ChIP与NGS相结合的方法，用于询问全基因组转录因子结合17 ，并且在某些情况下监测时间过程18,19,20的动态变化。例如，昼夜节律ChIP-seq研究已经显示出昼夜节律时钟组分和组蛋白标记物的基因组结合的高度编排的序列，其在整个〜24小时昼夜循环中驱动时间上精确的基因表达18 。 大多数可用的ChIP协议设计用于软组织（ 例如肝，脑等 ），并且很少已经出版用于硬组织，包括ke伸肌在富含纤维的骨骼肌中均匀化并分离高质量细胞核21是技术上的挑战，特别是对于需要交联的ChIP实验。在最近的肌肉ChIP研究22中 ，卫星细胞与肌纤维分离，并且通过涉及组织消化的延长的过程从两种细胞类型制备细胞核。在分离的核上进行甲醛交联之前，整个过程需要大约三个小时才能完成。虽然这种方法避免了交联肌纤维，这使得肌肉组织更有效地进行高效均匀化，并且能够产生高质量的细胞核，但是从组织收集到细胞核交联的显着的时间滞后引起了DNA变化的风险蛋白质相互作用。相比之下，大多数研究在实验治疗或组织收集后立即进行交联，以保存实时DNA-蛋白质n约束12 。在交联前核分离的第二个缺点是它排除了时间敏感的应用，例如通常以3-4小时间隔发生的昼夜样本采集。没有交联核，分离需要在解剖后立即进行，而交联样品可以在整个时间过程完成后一起处理，从而确保更大的实验一致性。 还报道了与未交联的骨骼肌分离核的其它方案。两项研究描述了使用梯度超速离心将细胞核与剩余的肌原纤维和细胞碎片分开23,24 。虽然蔗糖或胶体梯度超速离心对未交联的肌肉组织有效，但我们的实验表明，交联后，梯度超速离心不能将细胞从细胞分离出来ebris在梯度上。 因此，我们开发了一种使用交联的小鼠骨骼肌组织分离高质量核的方法。而不是梯度超速离心，我们设计了一种串联过滤方法来有效地将细胞核与碎片分离。经超声处理后，染色质样品成功应用于ChIP研究，显示BMAL1蛋白与靶启动子结合的昼夜节律模式。我们的方法可以广泛适用于肌肉组织的各种机械研究。

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			<td height="20" style="height:20px;width:216px;">Materials</td>
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			<td height="20" style="height:20px;width:216px;">Formaldehyde solution</td>
			<td style="width:143px;">Sigma Aldrich</td>
			<td style="width:119px;">F8775&#160;</td>
			<td></td>
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			<td height="20" style="height:20px;width:216px;">Glycine</td>
			<td style="width:143px;">Fisher Scientific</td>
			<td style="width:119px;">BP381-5</td>
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			<td height="20" style="height:20px;width:216px;">Trypan Blue solution (0.4%)</td>
			<td style="width:143px;">Fisher Scientific</td>
			<td style="width:119px;">15250061</td>
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			<td height="20" style="height:20px;width:216px;">RNase A</td>
			<td style="width:143px;">Sigma Aldrich</td>
			<td style="width:119px;">10109142001</td>
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			<td height="20" style="height:20px;width:216px;">Protease K</td>
			<td style="width:143px;">Sigma Aldrich</td>
			<td style="width:119px;">3115887001</td>
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			<td height="57" style="height:57px;width:216px;">Chicken Anti-BMAL1 antibody</td>
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			<td style="width:288px;">Generated in chicken (Cocalico Biologicals) against antigen aa 318-579, and IgY was affinity purified using the same antigen.&#160;</td>
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			<td height="20" style="height:20px;width:216px;">Chicken IgY Precipitating Resin</td>
			<td style="width:143px;">GenScript</td>
			<td style="width:119px;">L00405</td>
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			<td height="20" style="height:20px;width:216px;">Equipment</td>
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			<td height="38" style="height:38px;width:216px;">KINEMATICA&#160;Polytron PT2100 Benchtop Homogenizer</td>
			<td style="width:143px;">Fisher Scientific</td>
			<td style="width:119px;">08-451-178</td>
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			<td height="20" style="height:20px;width:216px;">15mL Dounce tissue grinder</td>
			<td style="width:143px;">Whearton</td>
			<td style="width:119px;">357544</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Falcon Cell Strainers 100&#181;m</td>
			<td style="width:143px;">Fisher Scientific</td>
			<td style="width:119px;">08-771-19</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Falcon Cell Strainers 70&#181;m</td>
			<td style="width:143px;">Fisher Scientific</td>
			<td style="width:119px;">08-771-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Falcon Cell Strainers 40&#181;m</td>
			<td style="width:143px;">Fisher Scientific</td>
			<td style="width:119px;">08-771-2</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">pluriStrainer 30 &#181;m</td>
			<td style="width:143px;">PluriSelect</td>
			<td style="width:119px;">43-50030-03</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">pluriStrainer 20 &#181;m</td>
			<td style="width:143px;">PluriSelect</td>
			<td style="width:119px;">43-50020-03</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">pluriStrainer 10 &#181;m</td>
			<td style="width:143px;">PluriSelect</td>
			<td style="width:119px;">43-50010-03</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Covaris S2 Focused-ultrasonicator</td>
			<td style="width:143px;">Covaris</td>
			<td style="width:119px;">Model S2</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Labquake&#160;</td>
			<td>Thermo Scientific</td>
			<td>C415110</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CCD Microscope Camera</td>
			<td>&#160;Leica Microsystems</td>
			<td>DFC3000 G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;"></td>
			<td style="width:143px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Reagent Kit</td>
			<td style="width:143px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">DNA extraction kit</td>
			<td style="width:143px;">Thermo Scientific</td>
			<td style="width:119px;">K0691</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;"></td>
			<td style="width:143px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Buffers</td>
			<td style="width:143px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="76">
			<td height="76" style="height:76px;width:216px;">All buffer components are discribed in the protocol. Each component was purchased from Sigma Aldrich</td>
			<td style="width:143px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

a filtration-based method of preparing high-quality nuclei from cross-linked skeletal muscle for chromatin immunoprecipitation

染色质免疫沉淀（ChIP）是确定蛋白质结合染色质DNA的有效方法。然而，富含纤维的骨骼肌由于技术上难以隔离高质量的细胞核而且对肌原纤维的污染最小，因此一直是ChIP的挑战。以前的方案尝试在交联之前纯化细胞核，这导致在延长的细胞核制备过程中DNA-蛋白质相互作用发生改变的风险。在目前的方案中，我们首先交联从小鼠收集的骨骼肌组织，并将组织切碎并超声处理。由于我们发现超速离心不能使用交联肌肉组织将原核与肌原纤维分开，我们设计了一个顺序的过滤程序，以获得没有显着肌原纤维污染的高质量细胞核。我们随后使用超声波仪制备了染色质，并且使用抗BMAL1抗体的ChIP测定显示强大的昼夜节律结合目的基因启动子的BMAL1模式。该过滤方案构成了将高质量细胞核与交联骨骼肌组织隔离的一种易于应用的方法，允许对昼夜节律和其他时间敏感性研究进行一致的样品处理。结合下一代测序（NGS），我们的方法可以部署用于骨骼肌功能的各种机械和基因组研究。

这里我们在组织收集后立即进行甲醛交联，以保持实时的DNA-蛋白质相互作用。然而，我们发现通常用于细胞核分离的蔗糖或胶体梯度23,24在分离细胞核和肌原纤维方面无效（数据未显示）。原因可能是交联赋予核和肌原纤维相似的重力。因此，我们开发了一个串联过滤方法，以有效地从核部分去除大的肌原纤维和其他碎屑。经过100μm过滤后，仍有较大的?...

Watch this Scientific Journal Video about A Filtration-based Method of Preparing High-quality Nuclei from Cross-linked Skeletal Muscle for Chromatin Immunoprecipitation at JoVE.com

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

我们提出了基于过滤的方案，以将高质量的细胞核与交联的小鼠骨骼肌分离，其中我们消除了超离心的需要，使其易于应用。我们显示从核制备的染色质适用于染色质免疫沉淀和可能的染色质免疫沉淀测序研究。

从交联骨骼肌制备高质量核的基于过滤的方法进行染色质免疫沉淀

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

Research

JoVE Journal

Biochemistry

A Facile and Efficient Approach for the Production of Reversible Disulfide Cross-linked Micelles

可逆二硫键交联胶束生产的简便和有效途径

Nanomedicine is an emerging form of therapy that harnesses the unique properties of particles that are nanometers in scale for biomedical application. ...

科研

生物化学

A Simple Fractionated Extraction Method for the Comprehensive Analysis of Metabolites, Lipids, and Proteins from a Single Sample

一种简单的分级提取方法，用于从单个样品中综合分析代谢物，脂质和蛋白质

Understanding of complex biological systems requires the measurement, analysis and integration of multiple compound classes of the living cell, usually ...

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

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

Activated Cross-linked Agarose for the Rapid Development of Affinity Chromatography Resins - Antibody Capture as a Case Study

活联甘蔗，用于亲和色谱树脂的快速发展 - 抗体捕获作为案例研究

The purification of monoclonal antibodies (mAbs) is commonly achieved by Protein A affinity chromatography, which can account for up to 25% of the overall ...

Quantification of Protein Interaction Network Dynamics using Multiplexed Co-Immunoprecipitation

使用多路复用共免疫沉淀对蛋白质相互作用网络动力学进行量化

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

Preparation of High-Quality Fermented Fish Product

优质发酵鱼产品的制备

This protocol provides a method for preparation of industrialized fermented fish product with sturgeon (Aquilaria sinensis) meat product. The procedures ...

Obtaining High-Quality Transcriptome Data from Cereal Seeds by a Modified Method for Gene Expression Profiling

通过基因表达分析的改良方法从谷物种子获取高质量的转录数据

The characterization of gene expression is dependent on RNA quality. In germinating, developing and mature cereal seeds, the extraction of high-quality ...

Preparing Lamellae from Vitreous Biological Samples Using a Dual-Beam Scanning Electron Microscope for Cryo-Electron Tomography

使用双光束扫描电子显微镜从玻璃体生物样品制备薄片进行冷冻电子断层扫描

Presented here is a protocol for preparing cryo-lamellae from plunge-frozen grids of Plasmodium falciparum-infected&#160;human erythrocytes, which could ...

Measurement of Protein Import Capacity of Skeletal Muscle Mitochondria

骨骼肌线粒体蛋白质导入能力的测定

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

Quantification of Subcellular Glycogen Distribution in Skeletal Muscle Fibers using Transmission Electron Microscopy

使用透射电子显微镜定量骨骼肌纤维中的亚细胞糖原分布

With the use of transmission electron microscopy, high-resolution images of fixed samples containing individual muscle fibers can be obtained. This ...