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

We thank Karyn Esser, Nobuya Koike and Noheon Park for helpful advice. This work was in part supported by NIH/NIGMS (R01GM114424) to S.-H.Y., and the Robert A. Welch Foundation (AU-1731) and NIH/NIA (R01AG045828) to Z.C.

Animal care was performed under Institutional Animal Care and Use Committee (IACUC) guidelines, and the procedures were conducted according to an animal protocol approved by the University of Texas Health Science Center at Houston.
1. Nuclei Isolation from Cross-linked Skeletal Muscle
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	<li>Weigh and mince hind limb skeletal muscle, isolated from approximately 20-week old C57BL/6 male mice, in ice-cold phosphate buffered saline (PBS) as described in detail previously22<...

<ol>
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Here we describe a robust method where cross-linked skeletal muscle tissues were used to isolate high-quality nuclei. Sequential filtration was carried out to effectively separate nuclei from debris, and ultrasonic acoustic energy from dish-shaped transducer sheared the chromatin for ChIP analysis. The results showed circadian time-specific binding of BMAL1 to target promoters.
ChIP can be employed to capture real-time protein occupancy on genomic DNA when cross-linking takes place. To take ad...

Skeletal muscle plays important roles in physiology and behavior. The multi-nucleated muscle fiber consists of myofibrils where actin and myosin form functional units called sarcomeres to generate contractile force. Skeletal muscle is also the largest metabolic organ in the body, accounting for &#62;80% postprandial glucose intake and regulating insulin response and metabolic homeostasis1,2. Muscle physiology and metabolism are closely regulated by the circadian clock, an intrinsic biological timer3,4,5,6. For example, the skeletal muscle-specific deletion of Bmal1, one of the core circadian clock components, resulted in insulin resistance and diminished glucose uptake in skeletal muscle, and the animals were found to develop type 2 diabetes7. In addition, skeletal muscle is also increasingly being appreciated as an endocrine organ8, secreting myokines to regulate systemic metabolism and physiology. Mechanistic studies are required to fully understand these regulatory functions in skeletal muscle.
ChIP is a powerful approach to delineate promoter recruitment of DNA binding proteins. ChIP was initially developed to identify nucleosome organization on chromatin DNA9,10. A variety of methods have since been reported to cross-link proteins and chromatin DNA using formaldehyde, dimethyl sulfate or ultraviolet irradiation (UV)11,12. Formaldehyde cross-linking is the most commonly used, preserving chromatin structure and DNA-protein interactions9,13,14. Cross-linked chromatin is shredded by sonication and immunoprecipitated with antibody against the particular DNA binding protein of interest15,16. In recent years, ChIP-sequencing (ChIP-seq), a method combining ChIP with NGS, has been developed to interrogate genome-wide transcription factor binding17, and in some cases to monitor dynamic changes over a time course18,19,20. For example, circadian ChIP-seq studies have revealed a highly orchestrated sequence of genomic binding of circadian clock components and histone markers, which drives temporally precise gene expression throughout the ~ 24 h circadian cycle18.
Most available ChIP protocols are designed for soft tissues (e.g. liver, brain, etc.), and very few have been published for hard tissues including skeletal muscle. It is technically challenging to homogenize fiber-rich skeletal muscle and isolate high-quality nuclei21, especially for ChIP experiments which require cross-linking. In a recent muscle ChIP study22, satellite cells were separated from myofibers, and nuclei were prepared from both cell types through a prolonged procedure involving tissue digestion. The entire process took approximately three hours to complete before formaldehyde cross-linking was performed on isolated nuclei. While this procedure avoided cross-linking muscle fiber, which makes muscle tissue even more refractory to efficient homogenization, and was able to produce high-quality nuclei, the significant time-lag from tissue collection to nuclei cross-linking incurs the risk of altered DNA-protein interaction. In contrast, most studies performed cross-linking immediately after experimental treatment or tissue collection in order to preserve the real-time DNA-protein binding12. A second drawback of nuclei isolation before cross-linking is that it precludes time-sensitive applications such as circadian sample collection which typically occurs at 3 - 4 h intervals. Without cross-linking the nuclei, the isolation needs to proceed immediately after dissection, whereas cross-linked samples can be processed together after the entire time course is completed, thus ensuring greater experimental consistency.
Other protocols for nuclei isolation from uncrosslinked skeletal muscle have also been reported. Two studies described the use of gradient ultracentrifugation to separate nuclei from remaining myofibrils and cell debris23,24. While sucrose or colloidal gradient ultracentrifugation is effective with uncrosslinked muscle tissues, our experiments revealed that after crosslinking, gradient ultracentrifugation failed to separate nuclei from cell debris on the gradient.
We therefore developed a procedure to isolate high-quality nuclei using cross-linked mouse skeletal muscle tissues. Rather than gradient ultracentrifugation, we devised a serial filtration method to effectively separate nuclei from debris. Following ultrasonication, the chromatin samples were successfully applied for ChIP studies which showed a circadian pattern of BMAL1 protein binding to target promoters. Our method can be broadly applicable to various mechanistic studies of muscle tissues.

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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>
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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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			<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>
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			<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>
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			<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>
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			<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>
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		<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>
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			<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>
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			<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>
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			<td height="20" style="height:20px;">Labquake&#160;</td>
			<td>Thermo Scientific</td>
			<td>C415110</td>
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			<td height="20" style="height:20px;">CCD Microscope Camera</td>
			<td>&#160;Leica Microsystems</td>
			<td>DFC3000 G</td>
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			<td height="20" style="height:20px;width:216px;"></td>
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			<td height="20" style="height:20px;width:216px;">Reagent Kit</td>
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			<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>
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			<td height="20" style="height:20px;width:216px;"></td>
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			<td height="20" style="height:20px;width:216px;">Buffers</td>
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		<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>
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</table>

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.

Here we performed formaldehyde cross-linking immediately after tissue collection to preserve real-time DNA-protein interaction. However, we found that sucrose or colloidal gradient, commonly used for nuclei isolation23,24, was not effective in separating nuclei from myofibrils (data not shown). The reason may be that cross-linking conferred similar gravity for nuclei and myofibrils. Therefore, we developed a serial filtration proc...

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

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.

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

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