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Developmental Biology

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

Published: July 7th, 2023

DOI:

10.3791/65215

1CNRS, Inserm, IGBMC UMR 7104- UMR-S 1258, Université de Strasbourg
* These authors contributed equally

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).

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, 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.

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 c....

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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'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.......

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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.......

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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&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.......

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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 'France Génomique' 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'US project (ANR 20-SFRI-0012) and EUR IMCBio (ANR-17-EURE-0023) under the ....

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

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