Name: Cistrome Analysis in Mouse Muscle Stem Cells
Uploaded: 2023-07-07T14:20:00
Description: Watch this Scientific Journal Video about Cistrome Analysis in Mouse Muscle Stem Cells at JoVE.com

We thank Anastasia Bannwarth for providing excellent technical assistance. We thank the IGBMC animal house facility, the cell culture, the Mouse Clinical Institute (ICS, Illkirch, France), the imaging, the electron microscopy, the flow cytometry, and the GenomEast platform, a member of the &#39;France G&#233;nomique&#39; consortium (ANR-10-INBS-0009).
This work of the Interdisciplinary Thematic Institute IMCBio, as part of the ITI 2021-2028 program of the University of Strasbourg, CNRS and Inserm, was supported by IdEx Unistra (ANR-10-IDEX-0002) and by SFRI-STRAT&#39;US project (ANR 20-SFRI-0012) and EUR IMCBio (ANR-17-EURE-0023) under the framework of the French Investments for the Future Program. Additional funding was delivered by INSERM, CNRS, Unistra, IGBMC, Agence Nationale de la Recherche (ANR-16-CE11-0009, AR2GR), AFM-T&#233;l&#233;thon strategic program 24376 (to D.D.), INSERM young researcher grant (to D.D.), ANR-10-LABX-0030-INRT, and a French State fund managed by the ANR under the frame program Investissements d&#39;Avenir (ANR-10-IDEX-0002-02). J.R. was supported by the Programme CDFA-07-22 from the Universit&#233; franco-allemande and Minist&#232;re de l&#39;Enseignement Sup&#233;rieur de la Recherche et de l&#39;Innovation, and K.G. by the Association pour la Recherche &#224; l&#39;IGBMC (ARI).

Mice were kept in an accredited animal house, in compliance with National Animal Care Guidelines (European Commission directive 86/609/CEE; French decree no.87-848) on the use of laboratory animals for research. Intended manipulations were submitted to the Ethical committee (Com&#39;Eth, Strasbourg, France) and to the French Research Ministry (MESR) for ethical evaluation and authorization according to the 2010/63/EU directive under the APAFIS number #22281.
1. Preparation of cell suspen...

Satellite Cell Isolation from Mouse Limb Muscles and Flow Cytometry Analysis

<ol>
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The present study reports a standardized, reliable, and easy-to-perform method for the isolation and culture of mouse satellite cells, as well as the assessment of transcriptional regulation by the CUT&#38;RUN method.
This protocol involves several critical steps. The first is muscle disruption and fiber digestion to ensure a high number of collected cells. Despite the increased enzyme concentration, more living cells were obtained than using Protocol 1. Satellite cells express a specific patt...

Skeletal striated muscles represent on average 40% of the weight of the total human body1. Muscle fibers exhibit a remarkable capacity for regeneration upon injury, which is described by the fusion of newly formed myocytes and the generation of new myofibers that replace the damaged ones2. In 1961, Alexander Mauro reported a population of mononuclear cells that he termed as satellite cells3. These stem cells express the transcription factor paired box 7 (PAX7), and are located between the basal lamina and the sarcolemma of muscle fibers4. They were reported to express the cluster of differentiation 34 (CD34; a hematopoietic, endothelial progenitor and mesenchymal stem cell marker), integrin alpha 7 (ITGA7; a smooth, cardiac and skeletal muscle marker), as well as the C-X-C chemokine receptor type 4 (CXCR4; a lymphocyte, hematopoietic, and satellite cell marker)5. In basal conditions, satellite cells reside in a particular microenvironment that keeps them in a quiescent state6. Upon muscle damage, they become activated, proliferate, and undergo myogenesis7. However, contributing only to a minor fraction of the total number of muscle cells, their genome-wide analyses are particularly challenging, especially under physiological settings (&#60;1% of total cells).
Various methods for chromatin isolation from satellite cells have been described, which involve chromatin immunoprecipitation followed by massive parallel sequencing (ChIP-seq) or cleavage under targets and tagmentation (CUT&#38;Tag) experiments. Nevertheless, these two techniques present some significant limitations that remain unchallenged. Indeed, ChIP-seq requires a high amount of starting material to generate enough chromatin, a large proportion of which is lost during the sonication step. CUT&#38;Tag is more appropriate for low cell number, but generates more off-target cleavage sites than ChIP-seq due to the Tn5 transposase activity. In addition, since this enzyme has a high affinity for open-chromatin regions, the CUT&#38;Tag approach might be preferentially used for analyzing histone modifications or transcription factors associated with actively transcribed regions of the genome, instead of silenced heterochromatin8,9.
Presented here is a detailed protocol that allows the isolation of mouse limb muscle satellite cells by FACS for cleavage under targets and release using nuclease (CUT&#38;RUN)10,11 analysis. The various steps involve the mechanical disruption of tissue, cell sorting, and nuclei isolation. The method&#39;s efficiency, regarding the preparation of a viable cell suspension, was demonstrated by performing CUT&#38;RUN analysis for covalent histone modifications and transcription factors. The quality of isolated cells makes the described method particularly attractive for preparing chromatin that captures the native genomic occupancy state faithfully, and is likely to be suitable for capturing the chromosome conformation in combination with high-throughput sequencing at specific loci (4C-seq) or at genome-wide levels (Hi-C).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5 mL microtube</td>
			<td>Eppendorf</td>
			<td>2080422</td>
			<td></td>
		</tr>
		<tr>
			<td>2 mL microtube</td>
			<td>Star Lab</td>
			<td>S1620-2700</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL tubes</td>
			<td>CORNING-FALCON</td>
			<td>352063</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL tubes</td>
			<td>Falcon</td>
			<td>352098</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-AR</td>
			<td>abcam</td>
			<td>ab108341</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-CD11b</td>
			<td>eBioscience</td>
			<td>25-0112-82</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-CD31</td>
			<td>eBioscience</td>
			<td>12-0311-82</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-CD34</td>
			<td>eBioscience</td>
			<td>48-0341-82</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-CD45</td>
			<td>eBioscience</td>
			<td>12-0451-83</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-CXCR4</td>
			<td>eBioscience</td>
			<td>17-9991-82</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-DMD</td>
			<td>abcam</td>
			<td>ab15277</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-H3K27ac</td>
			<td>Active Motif</td>
			<td>39133</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-H3K4me2</td>
			<td>Active Motif</td>
			<td>39141</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-ITGA7</td>
			<td>MBL</td>
			<td>k0046-4</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-PAX7</td>
			<td>DSHB</td>
			<td>AB_528428</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-TER119</td>
			<td>BD Pharmingen TM</td>
			<td>553673</td>
			<td></td>
		</tr>
		<tr>
			<td>Beads</td>
			<td>Polysciences</td>
			<td>86057-3</td>
			<td>BioMag&#174;Plus Concanavalin A</td>
		</tr>
		<tr>
			<td>Cell Strainer 100 &#181;m</td>
			<td>Corning&#174;&#160;</td>
			<td>431752</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Strainer 40 &#181;m</td>
			<td>Corning&#174;&#160;</td>
			<td>431750</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Strainer 70 &#181;m</td>
			<td>Corning&#174;&#160;</td>
			<td>431751</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge 1</td>
			<td>Eppendorf</td>
			<td>521-0011</td>
			<td>Centrifuge 5415 R</td>
		</tr>
		<tr>
			<td>Centrifuge 2</td>
			<td>Eppendorf</td>
			<td>5805000010</td>
			<td>Centrifuge 5804 R</td>
		</tr>
		<tr>
			<td>Chamber Slide System&#160;</td>
			<td>ThermoFischer</td>
			<td>171080</td>
			<td>Syst&#232;me Nunc&#8482; Lab-Tek&#8482; Chamber Slide</td>
		</tr>
		<tr>
			<td>Cleaning agent</td>
			<td>Sigma&#160;&#160;</td>
			<td>SLBQ7780V</td>
			<td>RNaseZAPTM</td>
		</tr>
		<tr>
			<td>Collagenase, type I&#160;</td>
			<td>Thermo Fisher</td>
			<td>17100017</td>
			<td>10 mg/mL</td>
		</tr>
		<tr>
			<td>Dispase&#160;</td>
			<td>STEMCELL technologies</td>
			<td>7913</td>
			<td>5 U/mL</td>
		</tr>
		<tr>
			<td>DynaMag&#8482;-2 Aimant</td>
			<td>Invitrogen</td>
			<td>12321D</td>
			<td></td>
		</tr>
		<tr>
			<td>Fixable Viability Stain</td>
			<td>BD Biosciences</td>
			<td>565388</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometer</td>
			<td>BD FACSAria&#8482; Fusion Flow Cytometer</td>
			<td>23-14816-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoromount G with DAPI</td>
			<td>Invitrogen</td>
			<td>00-4959-52</td>
			<td></td>
		</tr>
		<tr>
			<td>Genome browser&#160;</td>
			<td>IGV</td>
			<td></td>
			<td>http://software.broadinstitute.org/software/igv/</td>
		</tr>
		<tr>
			<td>Glycerol&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>G9012</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogel</td>
			<td>Corning&#174;&#160;</td>
			<td>354277</td>
			<td>Matrigel hESC qualified matrix</td>
		</tr>
		<tr>
			<td>Image processing software</td>
			<td>Image J&#174;</td>
			<td></td>
			<td>V 1.8.0</td>
		</tr>
		<tr>
			<td>Laboratory film</td>
			<td>Sigma-Aldrich</td>
			<td>P7793-1EA</td>
			<td>PARAFILM&#174; M</td>
		</tr>
		<tr>
			<td>Liberase LT</td>
			<td>Roche</td>
			<td>5401020001</td>
			<td></td>
		</tr>
		<tr>
			<td>Propyl gallate</td>
			<td>Sigma-Aldrich</td>
			<td>2370</td>
			<td></td>
		</tr>
		<tr>
			<td>Sequencer&#160;</td>
			<td>Illumina Hiseq 4000</td>
			<td>SY-401-4001</td>
			<td></td>
		</tr>
		<tr>
			<td>Shaking water bath</td>
			<td>Bioblock Scientific polytest 20</td>
			<td>18724</td>
			<td></td>
		</tr>
	</tbody>
</table>

an efficient protocol for cut&amp;run analysis of facs-isolated mouse satellite cells

Genome-wide analyses with small cell populations are a major constraint for studies, particularly in the stem cell field. This work describes an efficient protocol for the fluorescence-activated cell sorting (FACS) isolation of satellite cells from the limb muscle, a tissue with a high content of structural proteins. Dissected limb muscles from adult mice were mechanically disrupted by mincing in medium supplemented with dispase and type I collagenase. Upon digestion,&#160;the homogenate was filtered through cell strainers, and cells were suspended in FACS buffer. Viability was determined with fixable viability stain, and immunostained satellite cells were isolated by FACS. Cells were lysed with Triton X-100 and released nuclei were bound to concanavalin A magnetic beads. Nucleus/bead complexes were incubated with antibodies against the transcription factor or histone modifications of interest. After washes, nucleus/bead complexes were incubated with protein A-micrococcal nuclease, and chromatin cleavage was initiated with CaCl2. After DNA extraction, libraries were generated and sequenced, and the profiles for genome-wide transcription factor binding and covalent histone modifications were obtained by bioinformatic analysis. The peaks obtained for the various histone marks showed that the binding events were specific for satellite cells. Moreover, known motif analysis unveiled that the transcription factor was bound to chromatin via its cognate response element. This protocol is therefore adapted to study gene regulation in adult mice limb muscle satellite cells.

An Efficient Protocol for CUT&amp;RUN Analysis of FACS-Isolated Mouse Satellite Cells

Satellite cells from mouse skeletal muscles were isolated by combining the protocols of Gunther et al. (hereafter Protocol 1)12 and of Liu et al.23 (hereafter Protocol 2). Since non-digested muscle fibers were observed after digestion when using the concentration of collagenase and dispase proposed in Protocol 1, the quantity of enzymes was increased to improve muscle fiber dissociation, as described in steps 1.2.1 and 1.2.3. As indicated in Protocol 2, the samples were sub...

Watch this Scientific Journal Video about Cistrome Analysis in Mouse Muscle Stem Cells at JoVE.com

Cistrome Analysis in Mouse Muscle Stem Cells 

Presented here is an efficient protocol for the fluorescence-activated cell sorting (FACS) isolation of mouse limb muscle satellite cells adapted to the study of transcription regulation in muscle fibers by cleavage under targets and release using nuclease (CUT&amp;RUN).

Genome-wide analyses with small cell populations are a major constraint for studies, particularly in the stem cell field. This work describes an efficient ...

Since more than 20 years, our lab is interested in the pathophysiological role of nuclear receptors. To decipher their in vivo function, we engineered mouse model allowing the spatial and temporal control of their expression. In parallel, we developed cutting-edge techniques to investigate their molecular function in the mouse tissues.Our scientific interest recently led us to determine secosteroid and steroid receptor signaling in various tissues, including skeletal muscle. We have recently demonstrated that the androgen receptor in myofibers that differentiate cells of the skeletal muscle is instrumental in their contractile and metabolic functions. Our recent findings deliver a deeper understanding of skeletal muscle pathophysiological dynamics that is crucial to develop effective treatments for muscle-associated disorders.We developed this protocol to be able to perform cistrome analysis on muscle stem cells in vivo. All former protocols used in vitro modals, which completely dissociates the investigation from a whole organism context. In addition to being low-cost and time-efficient, this protocol provides an effective experimental setting to study transcription factor recruitment and chromatin landscape in skeletal muscle progenitor cells.Chromatin prepared by this protocol has provided the first genome-wide analysis of AR cistrome in satellite cells, and will facilitate future studies on gene regulation. Among many, our future aim would be to extend these investigations onto other oxosteroid receptors and explore their specific co-regulators in the various cell types involved in the regeneration process in skeletal muscle.