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

We thank all the staff of the IGBMC high throughput sequencing facility, a member of &#34;France G&#233;nomique&#34; consortium (ANR10-INBS-09-08) and all IGBMC general services in particular the staff of the IGBMC animal facility. This work was supported by grants from the CNRS, the INSERM, the AFM, the Ligue Nationale contre le Cancer, the French state fund through the ANR under the programme Investissements d&#39;Avenir labelled ANR-10-IDEX-0002-02, the Labex INRT ANR-10-IDEX-0001-02 and the ANR-AR2GR-16-CE11-009-01. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. S.J was supported by the Ligue Nationale contre le Cancer and the ANR-AR2GR-16-CE11-009-01, and V.U by the Minist&#232;re de l&#39;Enseignement et de la Recherche. ID is an &#39;&#233;quipe labellis&#233;e&#39; of the Ligue Nationale contre le Cancer.

Mice were kept in accordance with the institutional guidelines regarding the care and use of laboratory animals and in accordance with National Animal Care Guidelines (European Commission directive 86/609/CEE; French decree no.87-848). All procedures were approved by the French national ethics committee.
1. Isolation of Muscle Tissue
<ol>
	<li>Sacrifice one adult 6 to 8-week-old mouse by cervical dislocation. Sterlise the limb by rinsing it with 70% ethanol and dissect the hind limb muscles ...

<ol>
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Here we describe a novel protocol for preparing chromatin from adult mouse skeletal muscles and show that this chromatin is suitable for ChIP experiments that detect transcription factor binding and covalent histone modifications in the muscle fiber nuclei. This protocol involves several critical steps. The first is tissue disruption that can be performed either by dounce or by mechanical shearing. Mechanical shearing is faster and more reproducible, and is therefore the method of choice. Nevertheless, if no suitable app...

Chromatin immunoprecipitation (ChIP) coupled to quantitative polymerase chain reaction (qPCR) and high throughput sequencing (ChIP-seq) have become the methods of choice to study transcription and epigenetic regulation of gene expression in various tissues and cell-types1. This technique allows genome-wide profiling of covalent chromatin modifications, histone variant occupancy, and transcription factor binding2,3.
While performing ChIP from cultured cells is well established, ChIP from mammalian tissues remains more challenging. Preparing chromatin for ChIP involves several critical steps that need to be optimized for every tissue and cell type. Chromatin can be prepared from the cellular lysates of cultured cells or following purification of their nuclei. In the case of mammalian tissues, efficient lysis, formaldehyde fixation, and purification of nuclei are critical in order to ensure optimal recovery of the chromatin. Moreover, the choice of whether to fix the nuclei before or after their purification has to be experimentally determined. Despite these hurdles, ChIP has been successfully performed from tissues such as the liver, testis, or brain4,5,6. In the case of skeletal muscle, disruption of such a physically resistant tissue, which exhibits a high content of structural proteins, and isolation of its nuclei is particularly challenging. Given this specificity, protocols optimized for other tissues do not give satisfactory results for skeletal muscles.
Here we describe a protocol to isolate ChIP-grade chromatin from mouse skeletal muscle tissue that involves physically disrupting the tissue, formaldehyde fixation, and then the isolation of nuclei and sonication. The efficiency of this method to prepare chromatin suitable for ChIP from this tissue was demonstrated by performing ChIP-qPCR and ChIP-seq for various transcription factors, RNA polymerase II, and covalent histone modifications7.
This new technique is much faster than a previously reported protocol8 comprising long collagenase digestion steps during which time, changes in genomic localisation of transcription factors and alterations in gene expression may take place. The rapidity with which the nuclei are isolated and fixed makes the method described here particularly attractive to prepare chromatin that more faithfully captures the native genomic occupancy state. Moreover, although other methods for chromatin isolation or protein extraction from muscle tissue have have been described8,9,10 and used for ChIP-qPCR experiments on selected genes, no ChIP-seq data has been reported using them. The method reported here is suitable for transcription factor and histone modification ChIP-seq, and hence should be also suitable for 3/4C or HiC chromatin conformation capture applications.

<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

We describe an efficient and reproducible protocol for the preparation of chromatin from adult mouse skeletal muscle, a physically resistant tissue with a high content of structural proteins. Dissected limb muscles from adult mice are physically disrupted by mechanical homogenisation, or a combination of mincing and douncing, in a hypotonic buffer before formaldehyde fixation of the cell lysate. The fixed nuclei are purified by further cycles of mechanical homogenisation or douncing and sequential filtrations to remove cell debris. The purified nuclei can be sonicated immediately or at a later stage after freezing. The chromatin can be efficiently sonicated and is suitable for chromatin immunoprecipitation experiments, as illustrated by the profiles obtained for transcription factors, RNA polymerase II, and covalent histone modifications. The binding events detected using chromatin prepared by this protocol are predominantly those taking place in the muscle fiber nuclei despite the presence of chromatin from other fiber-associated satellite and endothelial cells. This protocol is therefore adapted to study gene regulation in the adult mouse skeletal muscle.

To isolate nuclei, we performed mechanical homogenization of the dissected and minced muscle tissue for either 15 or 45 s, at 18,000 and 22,000 rpm (see Table of Materials). In all conditions, nuclei could be separated from the tissue debris, but the yield was optimal using the lower speed (Figure 1A-B). Nuclei could also be prepared by douncing for 3 - 5 min (Figure 1A, and data not shown), but ...

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

A novel protocol for the preparation of chromatin from adult mouse skeletal muscle adapted to the study of gene regulation in muscle fibers by chromatin immunoprecipitation is presented.

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

The overall goal of this procedure is to prepare chromatin from adult mouse skeletal muscle. This method can help answer key questions in gene regulation and skeletal muscle fibers. The main advantage of this technique is that it allows preparation of ChIP-grade chromatin from mouse skeletal muscle.A physically resistant tissue with a high content of structural proteins. We first had the idea of this method when we were seeking to characterize the molecular mechanisms underlying gluco-articulated use muscle atrophy, which requires identification of gluco-articulated receptor, DNA-binding sites in mouse skeletal muscle fibers. Demonstrating the procedure will be Shilpy Joshi, a post-doc from our lab.To begin this procedure, recover frozen or freshly dissected hindlimb muscles. Subsequently, mince the muscles in a two-milliliter test tube, containing one milliliter of ice cold hypotonic buffer to a homogeneous preparation of small pieces, using fine scissors. Afterward, leave the tube shaking on a bench top agitator at four degrees Celsius for five to 10 minutes.Next, transfer the dissected tissue to a 14-milliliter round-bottom tube containing four milliliters of cold hypotonic buffer. Homogenize the minced tissue, using a mechanical tissue homogenizer for 15 to 30 seconds. Afterward, transfer the homogenate to a 15-milliliter tube.Add cold hypotonic buffer to result in 10 milliliters of the sample and fix the homogenate before proceeding with the next mouse. In this procedure, add formaldehyde to the sample to result in 1%final concentration. And then shake for 10 minutes at room temperature.Next, add glycine to result in a final concentration of 0.125 molar in each tube to stop fixation. And shake for five to 10 minutes at room temperature. After that, homogenize the fixed lysate, using a loose dounce.Then, transfer it to a 15-milliliter tube and centrifuge at 1000 g for five minutes at four degrees Celsius to obtain nuclei and cellular debris. After five minutes, remove the supernatant and re-suspend the pellet in five milliliters of fresh hypotonic buffer. Filter the lysate through a 70-micrometer cell strainer into a 50-milliliter tube.Using a pipette tip, wash the filter with one milliliter of cold hypotonic buffer. Following that, refilter the filtrate through a 40-micrometer cell strainer. Subsequently, transfer the filtrate to a 15-milliliter tube.And centrifuge at 1000 g for five minutes at four degrees Celsius to obtain the nuclear pellet. After discarding the supernatant, use one milliliter of hypotonic buffer to pool the three pellets and transfer to a 1.5 to two-milliliter tube. Centrifuge at 1000 g for five minutes.Next, assess the volume of the nuclear pellet by comparison with tubes containing a known volume of liquid and re-suspend it in sonication buffer up to three to four times of packed nuclear volume. Subsequently, sonicate the suspension for 10 to 15 minutes at four degrees Celsius, using a sonicator. Centrifuge the sonicated sample and transfer the chromatin supernatant to a fresh 1.5-milliliter tube.To de-crosslink, take 30 microliters of chromatin in a 1.5-milliliter test tube and add 20 microliters of five molar sodium chloride and 450 microliters of water. Complete the volume to 500 microliters and incubate at 65 degrees Celsius overnight. The next day, perform a treatment with one microliter of Proteinase K, 10 microliters of two molar tris at pH 6.8 and 10 microliters of 0.5 molar EDTA for one hour at 42 degrees Celsius.Then, perform a regular phenol-chloroform chloroform extraction and precipitate the DNA with one volume of sodium acetate and two volumes of 100%ethanol for one hour at negative 80 degrees Celsius or overnight at negative 20 degrees Celsius. Next, pellet the DNA by centrifugation at 13, 500 g for 15 minutes. Decant the supernatant and wash the pellet thoroughly with cold 70%ethanol and centrifuge again for five minutes.Then decant the supernatant and air-dry the pellet. After that re-suspend the pellet in the original volume of tris HCl EDTA buffer. Measure the DNA concentration in a spectrophotometer by absorbents at 260 nanometers, 280 nanometers.Subsequently, pipet 500 to 1000 nanograms of DNA together with a DNA-sized ladder on separate lanes of a 1.5%agarose gel. And perform electrophoresis to assess the fragment size of chromatin. To optimize, homogenization was performed for either 15 seconds or 45 seconds at two speeds, 18, 000 and 22, 000 RPM.In all conditions, nuclei could be separated from the tissue debris, but the yield was optimal using lower speed. Nuclei could also be prepared by douncing for three to five minutes. But mechanical homogenization is the method of choice as optimal yields of nuclei can be achieved in as little as 15 seconds, allowing an important gain of time, especially if multiple samples have to be processed.Using this protocol, up to 2.7 times 10 to the seven nuclei from 500 milligrams of tissue can be isolated to generate 100 micrograms of chromatin. Chromatin prepared in this way gives clean, high-quality results in ChIP sequencing experiments. As illustrated by the high levels of acetylated K27 of histone H3 and RNA polymerase 2 at the muscle-expressed Desmond locus.Specificity for muscle fiber is shown by low or absent signal at the Pecam1 and Vcam1 loci that are not expressed in muscle. While attempting this procedure, it is important to properly immerse the homogenizer probe in the sample to avoid frothing, to clean the probe between samples, and to optimize the chromatin fragmentation. Chromatin prepared in this way has been successfully used for ChIP's sake to map genome-wide transcription factor occupancy and should also be suitable for chromatin confirmation capture techniques.After it's development, this technique paved the way for research in the field of genomics, to explore gene regulation in skeletal muscle from mice. The procedure can also be applied to muscle from rat and biopsies from human patients. After watching this video, you should have a good understanding of how to use mechanical homogenization, followed by purification of formaldehyde-fixed nuclei to prepare chromatin from skeletal muscle for immunoprecipitation.Don't forget that working with formaldehyde, phenol, chloroform, and Proteinase K can be hazardous. Precautions such as wearing of gloves and glasses should be taken. Steps involving these reagents should be performed under a hood.

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