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.

Author Spotlight: Cistrome Analysis in Mouse Muscle Stem Cells 

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

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.

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

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 ...

an-efficient-protocol-for-cutrun-analysis-facs-isolated-mouse

Research

JoVE Journal

Developmental Biology

Preparation of Mouse Muscle Cell Suspension for Isolation of Satellite Cells by Fluorescence-Activated Cell Sorting

To begin, spray the sacrificed 10-week-old C57 black 6J male mouse thoroughly with 70%ethanol. Using forceps, remove the skin from the hindlimb and dissect all limb muscles surrounding the femur, tibia, and fibula. Place the harvested limb muscles into a two milliliter tube containing one milliliter of muscle isolation buffer.And using scissors, mince the harvested muscles. Transfer the minced muscle suspension into a 50 milliliter tube containing 18 milliliters of muscle isolation buffer. Close the tube tightly, seal it with laboratory film, and place it horizontally in a shaking water bath.After 30 minutes, add five milligrams of type I collagenase and maintain the tube for another 30 minutes. After pipetting the muscle suspension up and down 10 times, centrifuge and discard the supernatant, leaving five milliliters of the medium in the tube. Pipette the suspension onto the successive cell strainers and collect the flow-through into the 50 milliliter tube.Centrifuge the suspension and discard the supernatant until only 100 to 200 microliters remain in the tube. Resuspend the pellet in two milliliters of red blood cell lysis buffer and incubate on ice for three minutes. Centrifuge again and remove the supernatant from the tube using a 20 to 200 microliter pipette.Resuspend the pellet in 100 microliters of cold FACS buffer and place the tube on ice. After removing 10 microliters of cell suspension for the negative control, centrifuge the remaining 90 microliters and discard the supernatant before incubating the cells with 400 microliters of fixable viability stain diluted in serum-free DMEM for 15 minutes at room temperature. After centrifugation, add 100 microliters of FACS buffer and gently invert the tube thrice.Centrifuge again and add 100 microliters of primary antibody mixture into the tube. After 30 minutes of incubation in the dark, centrifuge and discard the supernatant. Then add 500 microliters of PBS to wash the cells.Centrifuge again and remove the supernatant before resuspending the pellet in 500 microliters of FACS buffer. Transfer the suspension to a five milliliter tube. Briefly vortex the cell suspension before processing it on a flow cytometer.Collect the selected cells in the five milliliter coated tube containing FACS buffer. 75%of the mononucleated cells identified based on their size and granularity were negative for the fixable viability stain marker leading to an average of 34.3%of live cells. About 3%of single living cells were negative for monocyte, endothelial, leukocyte, and erythroid specific markers.These negative cells were then selected according to their expression of CD34 and ITGA7 markers. A final gating for CXCR4 was performed to select putative satellite cells. And from the CD34-positive ITGA7-positive cells, 80%were found to be positive for CXCR4.

Cleavage Under Targets and Release Using Nuclease Analysis of FACS-Sorted Mouse Satellite Cells

Centrifuge the FACS-isolated satellite cells and discard the supernatant. Wash the cells with one milliliter of PBS, centrifuge, and incubate them in one milliliter cold nuclear extraction buffer on ice for 20 minutes. Add 20 microliters of Concanavalin A coated magnetic beads into a 1.5 milliliter tube containing 850 microliters of cold binding buffer.Then using a magnetic rack, wash the beads twice with one milliliter of cold binding buffer and allow the beads to accumulate at the side of the tube on the rack for five minutes before gently resuspending them in 300 microliters of cold binding buffer. Next, centrifuge the nuclei and resuspend them gently in 600 microliters of nuclear extraction buffer. Then mix 600 microliters of extracted nuclei with 300 microliters of Concanavalin A bead slurry and incubate at four degrees Celsius for 10 minutes.After removing the supernatant using a magnetic rack, resuspend the bead bound nuclei with one milliliter of cold blocking buffer. Again, remove the supernatant and wash the bead bound nuclei twice with one milliliter of cold wash buffer. Separate the supernatant, gently resuspend the nucleus bead complexes with a specific primary antibody and incubate at four degrees Celsius overnight with gentle agitation.The next day, remove the supernatant and wash the bead bound nuclei before resuspending in 100 microliters of cold wash buffer. Add 100 microliters of diluted protein A-micrococcal nuclease to the 100 microliters of bead bound nuclei and incubate at four degrees Celsius for one hour with agitation. After incubation, remove the supernatant and wash the bead bound nuclei twice before resuspending them in 150 microliters of cold wash buffer.Next, add three microliters of 100 millimolar calcium chloride, mix quickly by flicking and incubate on ice for 30 minutes. Stop the reaction by adding 150 microliters of stop buffer and incubate at 37 degrees Celsius for 20 minutes. Centrifuge the samples and transfer the supernatant to a new microcentrifuge tube, discarding the pellet and beads.Then add three microliters of 10%sodium dodecyl sulfate and 2.5 microliters of 20 milligrams per milliliter proteinase K.Mix by inversion and incubate for 10 minutes at 70 degrees Celsius. Then add 300 microliters of phenol:chloroform:isoamyl alcohol and vortex before transferring to two milliliter phase lock tubes. Centrifuge and add 300 microliters of chloroform to the same tube.Centrifuge again and collect the supernatant into a 1.5 milliliter tube. Next, add one microliter of glycogen, then 750 microliters of 100%ethanol, and allow the DNA to precipitate overnight at minus 20 degrees Celsius. The next day, pellet the DNA by centrifugation, then wash the pellet with one milliliter of 100%ethanol and centrifuge twice before removing residual ethanol with a pipette.Air dry the pellet for five minutes and resuspend it in 25 microliters of tris-EDTA buffer. The H3KME2 and H3K27AC were enriched around the TSS of PAC7, ITGA7, LAM2, CXCR4, and VCAM1. However, H3K4ME2 and H3K27AC were not enriched at those of ITGAM, PTPRC, PECAM, CKM, or MHY3.Almost no signal was obtained with the IgG sample. HOMER known motif analysis AR peaks and satellite cells and percent targeted regions are shown. Seeker analysis unveiled almost 500 peaks with a peak score higher than 50 with more than 200 of them with a score higher than 100.

1.5K Views.  Université de Strasbourg.  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&RUN).

Satellite Cell Isolation from Mouse Limb Muscles and Flow Cytometry Analysis

Summary