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 (Gastrocnemius, Tibialis Anterior, Quadriceps) using fine point scissors and forceps. 
	NOTE: One mouse should yield around 500 mg of tissue.</li>
	<li>Mince the muscles in a 2 mL test tube containing 1 mL of ice-cold hypotonic buffer to a homogenous preparation of small (&#60;2 - 3 mm3) pieces using fine scissors and leave the tube shaking on a bench top agitator at 4 &#176;C for 5 - 10 min.</li>
</ol>
2. Tissue Lysis
NOTE: Please refer to Table 1 for all buffer compositions.
<ol>
	<li>Transfer the homogenate to a 14 mL round-bottom tube and resuspend in 5 mL cold hypotonic buffer (EDTA-free protease inhibitor cocktail and PMSF).
	<ol>
		<li>Homogenise the minced muscle tissue using a loose dounce (20 - 30 strokes within 3 min) or a mechanical tissue homogenizer for 15 - 30 s in round-bottom 14 mL tubes. 
		NOTE: The efficiency of lysis can be assessed at this stage by light microscopy, and if required, additional disruption can be performed.</li>
	</ol>
	</li>
	<li>Transfer the homogenate to 15 mL tubes and fill volume to 10 mL using cold hypotonic buffer. Fix the homogenate as described below before proceeding with the next mouse.</li>
</ol>
3. Fixation
<ol>
	<li>Add formaldehyde to 1% final concentration and shake for 10 min at room temperature.</li>
	<li>Add glycine to a final concentration of 0.125 M in each tube to stop fixation and shake for&#160;5 - 10 min at room temperature.</li>
</ol>
4. Nuclei Preparation
NOTE: Please refer to Table 1 for all buffer compositions.
<ol>
	<li>Homogenise the fixed lysate using a loose dounce (5 - 10 strokes).
	<ol>
		<li>Transfer to a 15 mL tube and centrifuge at 1,000 x g for 5 min at 4 &#176;C to obtain nuclei and cellular debris.</li>
	</ol>
	</li>
	<li>Remove the supernatant and resuspend the pellet in fresh 5 mL hypotonic buffer. Filter the lysate through a 70 &#181;m cell strainer into a 50 mL tube. Re-filter the filtrate through a 40 &#181;m cell strainer.</li>
	<li>Transfer the filtrate to a 15 mL tube and centrifuge at 1,000 x g for 5 minutes at 4 &#176;C to obtain the nuclear pellet. 
	NOTE: At this stage, the nuclei can be immediately sonicated, or snap frozen as a dry pellet in liquid nitrogen and stored at -80 &#176;C.</li>
</ol>
5. Sonication
<ol>
	<li>Assess the volume of the nuclear pellet (around 50 &#181;L for both mechanical and dounce homogenisation) and resuspend it in sonication buffer up to 3 to 4 times of packed nuclear volume and sonicate for 10 - 15 min at 4 &#176;C using a sonicator. 
	NOTE: The chromatin can be analysed immediately as described below (6) or frozen and stored at -80 &#176;C.</li>
</ol>
6. Assessing the Chromatin Fragment Size following Sonication
NOTE: Please refer to Table 1 for all buffer compositions.
<ol>
	<li>To de-crosslink, take 30 &#181;L of chromatin in a 1.5 mL test tube and add 20 &#181;L 5 M NaCl. Complete the volume to 500 &#181;L and incubate at 65 &#176;C overnight.
	<ol>
		<li>The next day, perform a treatment with 1 &#181;L proteinase K (20 mg/mL stock), 10 &#181;L 2 M Tris pH 6.8, and 10 &#181;L of 0.5 M EDTA for 1 h at 42 &#176;C. Perform a phenol-chloroform/chloroform extraction and precipitate the DNA with 1 volume of sodium acetate (3 M) and 2 volumes of 100% ethanol for one h at -80 &#176;C or overnight at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Pellet the DNA by centrifugation at 13,500 x g for 15 min. Decant the supernatant and wash the pellet thoroughly with cold 70% ethanol and centrifuge again for 5 min. Decant the supernatant and air-dry the pellet.</li>
	<li>Resuspend the pellet in the original volume (30 &#181;L) of TE buffer. Measure the DNA concentration in a spectrophotometer by absorbance at 260 nm/280 nm. Pipette 500 - 1,000 ng of DNA together with a DNA size ladder on separate lanes of a 1.5% agarose gel and perform electrophoresis to assess the fragment size of chromatin. 
	NOTE: Fragment size should be between 200 - 500 base pairs, and there should be no detectable high molecular weight fragments corresponding to non- or poorly-fragmented DNA. If required, the chromatin solution (step 5) can be re-sonicated for a longer time and then re-analysed as described above until the optimum size is obtained.</li>
</ol>
7. Chromatin Immunoprecipitation (ChIP)
NOTE: Please refer to Table 1 for all buffer compositions.
<ol>
	<li>Block the Protein G sepharose beads by aliquoting 1 mL of the 50% slurry in ethanol. Pellet the beads by centrifugation at 400 x g for 1 min in a benchtop centrifuge and wash with 1 mL of TE buffer centrifuge at 400 x g and repeat the wash.
	<ol>
		<li>After the second centrifugation, re-suspend in 1 mL of ChIP dilution buffer with 25 &#181;L of BSA (stock 20 mg/mL) and 20 &#181;L yeast tRNA (stock 10 mg/mL). Block the beads for at least 2 h prior to use by rotation (40 - 60 rpm) at 4 &#176;C.</li>
		<li>Dilute 50 &#181;g of chromatin (step 5) 8 to 10 times using ChIP dilution buffer. Add 50 &#181;L of blocked bead slurry and incubate for 2 h rotating (40-60 rpm) at 4&#176;C. Centrifuge at 400 x g at 4 &#176;C to obtain pre-cleared chromatin and transfer it to a fresh tube.</li>
	</ol>
	</li>
	<li>For immunoprecipitation, add the appropriate amount of primary antibody (e.g., 1-2 &#181;g per 10 &#181;g of chromatin) and incubate overnight with rotation at 4 &#176;C. 
	NOTE: The optimal amount of antibody may be recommended by the supplier or can be determined empirically by performing ChIP-qPCR with different quantities of antibody until optimum enrichment is observed. Also Protein G sepharose may be replaced by Protein A sepharose depending on the subtype of antibody used.</li>
	<li>Add the blocked beads the next day and incubate for 1 h with rotation (40 - 60 rpm) at 4 &#176;C. Centrifuge at 400 x g for 20 - 30 s to pellet the beads, remove the supernatant, and start the ChIP washes.</li>
	<li>ChIP washes: Perform the following washes with the buffers: once with Low Salt Buffer, and then twice with High Salt Buffer, twice with LiCl Buffer, and finally twice with TE Buffer.
	<ol>
		<li>Perform each wash for 10 min with rotation (40 - 60 rpm) at 4 &#176;C and pellet the beads between each wash by centrifugation at 400 x g for 20 - 30 s in a bench-top centrifuge.</li>
	</ol>
	</li>
	<li>For elution, remove the last wash and resuspend the beads in 250 &#181;L elution buffer (freshly prepared) for 15 min at room temperature while shaking.</li>
	<li>Centrifuge at 400 x g and collect the eluate in a fresh tube. Perform this step twice and then de-crosslink the eluate overnight with 20 &#181;L NaCl 5 M and 1 &#181;L RNase A (10 mg/mL), treat with proteinase K, and phenol-chloroform/chloroform extract as in Step 6.</li>
	<li>Resuspend in 50 &#181;L of TE buffer and use aliquots for ChIP-qPCR as per standard protocols15 to check the quality of the ChIP.</li>
</ol>

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	<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>

The authors declare that they have no competing financial interests.

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 apparatus is available, disruption can also be performed by douncing, where assessment of the efficiency of lysis under the microscope is recommended before proceeding to the fixation step. Fixation of whole-tissue lysates, rather than fixation of isolated nuclei, allowed more reproducible sonication in smaller volumes and for short durations. We also chose to fix the lysates after disruption rather than fix the tissue before disruption, as described by Thomas et al.10 Pre-fixing the tissue resulted in reduced efficiency of homogenisation. This protocol based on mechanical shearing is also faster than other proposed solutions, such as collagenase digestion, which involve prolonged incubation of the dissected tissue at 37 &#176;C8. During this period, changes in gene expression and transcription factor binding can occur. Fixing the nuclei as fast as possible is therefore essential to more faithfully capture the normal physiological state.
A second critical step is the purification of nuclei from the fixed lysate. For this, sequential filtering of the fixed lysate solution, first through a 70 &#181;m cell strainer to eliminate the bigger debris and then through a 40 &#181;m cell strainer to remove the fine debris, avoids clogging the strainers and maximizes the yields of nuclei obtained. Once purified by filtration, nuclei are sonicated and the DNA fragment size determined after de-crosslinking. A typical preparation from the two hind limbs of one mouse yields 100 &#181;g of chromatin. Pooling the lysates from up to 3 mice may improve the yield by reducing the losses during preparation. This procedure is efficient as it allows the recovery of up to 75% of the genomic DNA as chromatin (data not shown).
ChIP experiments designed to assess transcription factor binding and epigenetic modifications demonstrated the suitability of this protocol for studying gene regulation in the muscle fiber nuclei. Strong and robust ChIP-seq signals for Pol II and H3K27ac were generated from the chromatin, allowing identification of muscle fiber identity genes by their distinctive H3K27ac and Pol II profiles7. The much higher levels of H3K27ac and Pol II on genes strongly expressed in fiber nuclei, compared to genes expressed in satellite or endothelial cells, showed that the signal came predominantly from the fiber nuclei. This was also confirmed by the loss of Tead4 ChIP signal using chromatin from mice where it was specifically inactivated in muscle fibers. Nevertheless, weaker, but clearly above background signals, for satellite cell markers such as Vcam1, Pax7 (data not shown) can be detected showing that chromatin from these cells is also present.&#160;We anticipate that our protocol should be suitable for other techniques that use formaldehyde-fixed chromatin such as 3C/4C and HiC chromatin conformation capture techniques. Use of our protocol for combining ChIP-seq for transcription factors and chromatin modifications with chromatin conformation capture should in the future allow a detailed understanding of gene regulatory mechanisms in muscle fiber and how these may be regulated or upset by physiological stimuli, such as exercise, fasting, a high fat diet, denervation, or in disease, by using chromatin prepared from genetically modified mice reproducing diseases like X-linked centronuclear myopathy14.
In summary, this low-cost and time-efficient protocol allows the isolation of muscle fiber nuclei that can be sonicated immediately or frozen at -80 &#176;C for ulterior use. Use of chromatin prepared by this protocol has provided the first genome wide ChIP-seq data from muscle fiber7 and will facilitate future studies on gene regulation in this tissue.

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 mechanical homogenization is the method of choice, as optimal yields of nuclei can be achieved in as little as 15 s, allowing an important gain of time especially if multiple samples have to be processed (Figure 1B). Using this protocol, up to 2.7 x 107 nuclei from 500 mg of tissue (equivalent to 1 mouse&#39;s tissue) can be isolated to generate 100 &#181;g of chromatin.
To optimize chromatin fragmentation from fixed nuclei, we sonicated for 5 to 20 min. Under the settings (see Table of Materials), a 10 min sonication was optimal to generate chromatin with an average fragment size of 250 bp (Figure 2). This step should be optimized experimentally taking into account the type of sonicator used.
To assess the quality of the muscle chromatin prepared by the protocol described above, ChIP-qPCR and ChIP-seq experiments were performed against transcription factors, a histone modification or RNA polymerase II (Pol II). ChIP-qPCR against the glucocorticoid receptor (GR) in the presence or absence of dexamethasone (Dex) treatment revealed strong Dex-dependent GR enrichment at regulatory elements of the Redd1 and Murf1 genes that are known to be Dex and GR regulated in muscle fibers11, while no signal was seen at the testis specific Prm1 promoter used as a negative control (Figure 3A).
ChIP-qPCR against acetylated lysine 27 of histone H3 (H3K27ac), a covalent modification found at active enhancers and promoters, showed strong enrichment at the Desmin (Des) promoter that is active in muscle fibers, compared to a negative intergenic control region (Figure 3B). H3K27ac ChIP-seq revealed a high level of this mark throughout the Des locus, typical of a &#39;super-enhancer&#39; regulated tissue identity gene12 (Figure 4A). Similarly, Pol II ChIP-seq showed high levels of transcribing Pol II at this locus.
ChIP-qPCR for transcription factor Tead4, which plays an important role in myogenic differentiation13,7, revealed its enrichment on previously described binding sites at the Ccnd1 and Ifrd1 genes (Figure 3C). Importantly, enrichment was reduced using chromatin prepared from mice where Tead4 was selectively inactivated in muscle fibers (Tead4skm-/-)7 (Figure 3C). Analyses of ChIP-seq data for both Tead1 and Tead4 showed their binding to a site in the promoter of the Kdm5a gene using chromatin from wild-type muscle, whereas the Tead4 signal, but not that of Tead1, H3K27ac, or Pol II, was lost using the chromatin from muscles of the Tead4skm-/- mice (Figure 4B). Similarly, Tead1 and Tead4 binding to a regulatory element downstream of the Adssl1 gene is seen with chromatin from wild-type muscle, whereas Tead4 binding was selectively lost using chromatin from the Tead4skm-/- muscle (Figure 4C). These observations demonstrate that the Tead4 signal seen in the ChIP-seq data came from binding in the muscle fiber.
To determine the contribution of other cell types associated with the muscle fiber to the ChIP-seq results, we assessed the H3K27ac and Pol II signals at the Vcam1 gene that is active in muscle satellite cells and Pecam1, a marker of endothelial cells of blood vessel that irrigate the muscles. Compared to the high levels seen at the Des or Adssl1 genes, the levels of H3K27ac and Pol II seen at both the Vcam1 and Pecam1 genes were much lower (Figure 4A and Figure 4C). The large differences in signal for muscle fiber expressed genes compared to those expressed in associated tissues indicated that the signal seen in ChIP from the chromatin prepared using the protocol described above comes predominantly from binding events in the muscle fiber.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56504/56504fig1.jpg" /> 
Figure 1: Purification of nuclei from adult mouse muscle. (A) Tissue lysates obtained by mechanical homogenisation (18,000 rpm, 45 s) and dounce homogenization (30 strokes/3 min) were stained with trypan blue and observed under a digital microscope and images were captured at 20X&#160;magnification. Scale bar = 100 &#181;m. (B) Number of nuclei obtained from 500 mg of tissue (equivalent of 1 mouse) after preparation under the indicated conditions, 18,000 and 22,000 rpm for 15 and 45 s or 3 min of douncing. Error bars represent mean &#177; S.E.M. for n = 3 mice for mechanical homogenization, and n = 3 mice for douncing. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56504/56504fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56504/56504fig2.jpg" /> 
Figure 2: Verification of chromatin fragment size. Isolated nuclei were sonicated for 5, 10, 15 or 20 min as indicated. After de-crosslinking and purification, the DNA fragment size was assessed by agarose gel electrophoresis in the presence of ethidium bromide. M is DNA size marker. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56504/56504fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56504/56504fig3.jpg" /> 
Figure 3: Use of chromatin for ChIP-qPCR. (A) Mice were subjected to intra-peritoneal injections of vehicle or dexamethasone (10 mg/kg) and after 1.5 h, chromatin was prepared from hind limb muscles. The enrichment in ChIP at GR target genes Redd1 and Murf1, compared to the negative control protamine promoter (Prm1), is indicated as a % of input precipitated. (B) ChIP was performed for H3K27ac and IgG control antibodies and analysed at the Desmin promoter and compared to an intergenic negative control region. (C) Tead4 ChIP was performed on muscle chromatin obtained from wild type and muscle-specific Tead4 knockout mice (Tead4skm-/-) and analysed at Tead4 binding sites in the Ccnd1 and Ifrd1 promoters, compared to the control intergenic region. In all experiments, chromatin from 3 mice was pooled and error bars represent mean &#177; S.E.M in a Student&#39;s T-test for three technical replicates. P value &#60;0.001**, &#60;0.0001 ***; NS, non-significant. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56504/56504fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56504/56504fig4.jpg" /> 
Figure 4: Genomic profiles of muscle fiber chromatin. (A) Screenshots from the University of California at Santa Cruz genome browser of the indicated loci illustrating the much higher H3K27ac and Pol II levels at the Desmin gene, highly expressed in muscle fiber, compared to the Vcam1 and Pecam1 genes expressed in satellite cells and endothelial cells, respectively. (B) UCSC screenshots of the indicated loci illustrating binding of Tead1 and Tead4, as well as H3K27ac and Pol II, in chromatin from wild-type (WT) muscle compared to that from Tead4skm-/- (MT) muscle, where selective loss of Tead4 binding is observed. The ChIP-seq data illustrated is available as GSE82193 in the GEO database. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56504/56504fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Buffer Name</td>
			<td colspan="3">Composition</td>
		</tr>
		<tr>
			<td rowspan="6">Hypotonic buffer</td>
			<td colspan="3">10 mM HEPES-KOH (pH 7.3)</td>
		</tr>
		<tr>
			<td colspan="3">10 mM KCl</td>
		</tr>
		<tr>
			<td colspan="3">5 mM MgCl2</td>
		</tr>
		<tr>
			<td colspan="3">0.1% NP-40</td>
		</tr>
		<tr>
			<td colspan="3">0.1 mM PMSF (freshly added)</td>
		</tr>
		<tr>
			<td colspan="3">Protease Inhibitor cocktail (PIC) 1x (freshly added)</td>
		</tr>
		<tr>
			<td rowspan="8">Sonication buffer</td>
			<td colspan="3">1% SDS</td>
		</tr>
		<tr>
			<td colspan="3">10 mM EDTA</td>
		</tr>
		<tr>
			<td colspan="3">20 mM Tris-HCl pH 8</td>
		</tr>
		<tr>
			<td colspan="3">150 mM NaCl</td>
		</tr>
		<tr>
			<td colspan="3">0.1% Sodium Deoxycholate</td>
		</tr>
		<tr>
			<td colspan="3">1% Triton X-100</td>
		</tr>
		<tr>
			<td colspan="3">Freshly added 0.1 mM PMSF</td>
		</tr>
		<tr>
			<td colspan="3">PIC 1x</td>
		</tr>
		<tr>
			<td rowspan="7">ChIP Dilution buffer</td>
			<td colspan="3">0.01% SDS</td>
		</tr>
		<tr>
			<td colspan="3">1.1% Triton X-100</td>
		</tr>
		<tr>
			<td colspan="3">1.2 mM EDTA</td>
		</tr>
		<tr>
			<td colspan="3">16.7 mM Tris HCl pH8</td>
		</tr>
		<tr>
			<td colspan="3">167 mM NaCl</td>
		</tr>
		<tr>
			<td colspan="3">Freshly added 0.1 mM PMSF</td>
		</tr>
		<tr>
			<td colspan="3">PIC 1x</td>
		</tr>
		<tr>
			<td rowspan="5">Low Salt buffer</td>
			<td colspan="3">0.1% SDS</td>
		</tr>
		<tr>
			<td colspan="3">1% Triton X-100</td>
		</tr>
		<tr>
			<td colspan="3">500 mM EDTA</td>
		</tr>
		<tr>
			<td colspan="3">20 mM Tris HCl pH8</td>
		</tr>
		<tr>
			<td colspan="3">150 mM NaCl</td>
		</tr>
		<tr>
			<td rowspan="5">High Salt buffer</td>
			<td colspan="3">0.1% SDS</td>
		</tr>
		<tr>
			<td colspan="3">1% Triton X-100</td>
		</tr>
		<tr>
			<td colspan="3">500 mM EDTA</td>
		</tr>
		<tr>
			<td colspan="3">20 mM Tris HCl pH8</td>
		</tr>
		<tr>
			<td colspan="3">500 mM NaCl</td>
		</tr>
		<tr>
			<td rowspan="4">LiCl Buffer</td>
			<td colspan="3">250 mM LiCl</td>
		</tr>
		<tr>
			<td colspan="3">1% NP-40</td>
		</tr>
		<tr>
			<td colspan="3">1 mM EDTA</td>
		</tr>
		<tr>
			<td colspan="3">10 mM Tris HCl pH 8</td>
		</tr>
		<tr>
			<td rowspan="2">Elution buffer</td>
			<td colspan="3">1% SDS</td>
		</tr>
		<tr>
			<td colspan="3">100 mM NaHCO3</td>
		</tr>
		<tr>
			<td>Tris-EDTA (TE) buffer</td>
			<td colspan="3">50 mM Tris HCl pH 8</td>
		</tr>
		<tr>
			<td></td>
			<td colspan="3">1 mM EDTA</td>
		</tr>
	</tbody>
</table>
Table 1: Buffer compositions.

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Biochemistry

8.4K Views.  Institut de Génétique et de Biologie Moléculaire et Cellulaire. 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.

Video: 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.

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

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.

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.

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.

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.

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.

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.

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.

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.

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.

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