An Efficient Protocol for CUT&RUN Analysis of FACS-Isolated Mouse Satellite Cells
July 7th, 2023
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).
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.
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Overview
This article presents a protocol for the fluorescence-activated cell sorting (FACS) isolation of mouse limb muscle satellite cells. This method is adapted for studying transcription regulation in muscle fibers using the CUT&RUN technique.
Key Study Components
Area of Science
- Neuroscience
- Cell Biology
- Molecular Biology
Background
- The lab has focused on the role of nuclear receptors for over 20 years.
- Mouse models have been engineered for spatial and temporal control of receptor expression.
- Techniques have been developed to investigate molecular functions in mouse tissues.
- Recent studies have explored steroid receptor signaling in skeletal muscle.
Purpose of Study
- To understand the role of the androgen receptor in skeletal muscle.
- To investigate the contractile and metabolic functions of myofibers.
- To develop effective treatments for muscle-associated disorders.
Methods Used
- Fluorescence-activated cell sorting (FACS)
- Cleavage under targets and release using nuclease (CUT&RUN)
- Mouse model engineering
- Analysis of receptor signaling pathways
Main Results
- The androgen receptor is crucial for myofiber function.
- Findings enhance understanding of skeletal muscle dynamics.
- Research contributes to potential treatments for muscle disorders.
Conclusions
- The study provides insights into transcription regulation in muscle cells.
- It highlights the importance of nuclear receptors in muscle physiology.
- Future research may lead to novel therapeutic strategies.
Frequently Asked Questions
Executive Industry Relevance
Genome-wide chromatin profiling in rare stem cell populations is a critical bottleneck for target validation and mechanistic de-risking in muscle biology. This protocol enables high-confidence mapping of transcription factor binding and histone modifications in FACS-isolated mouse satellite cells, directly supporting early discovery and translational research. The approach advances predictive confidence for gene regulation studies in disease-relevant muscle stem cell systems.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables direct interrogation of transcription factor recruitment in primary muscle stem cells.
- Supports functional target validation by mapping chromatin landscapes in vivo.
- Facilitates mechanistic de-risking for nuclear receptor signaling pathways.
- Provides genome-wide data to inform portfolio triage and prioritization.
Screening & Assay Development
- Delivers validated chromatin profiling workflows for rare cell populations.
- Standardizes sample preparation and immunostaining for reproducible outputs.
- Generates quantitative readouts of transcription factor and histone mark occupancy.
- Prepares high-quality libraries suitable for downstream sequencing and analysis.
Translational & Preclinical Research
- Aligns chromatin state mapping with disease-relevant muscle regeneration models.
- Enables continuity from discovery-stage findings to preclinical validation in vivo.
- Supports identification of regulatory elements and co-regulators in muscle repair.
- Provides a platform for future studies on additional nuclear receptor families.
Pipeline & Workflow Integration
This protocol bridges early discovery and preclinical research by enabling cistrome analysis in freshly isolated satellite cells, supporting both target validation and mechanistic studies.
- Discovery Biology: Facilitates hypothesis testing on transcription factor function in native muscle stem cells.
- Screening: Establishes reproducible, quantitative chromatin profiling for rare cell types.
- Analytics: Provides genome-wide binding and histone modification data for comparative analysis.
- Translational Research: Connects chromatin state changes to muscle regeneration and disease models.
- Enterprise Reuse: Adaptable to other transcription factors and cell types for broader R&D impact.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence in gene regulation and target engagement.
- Operational Value: Streamlines chromatin profiling in low-abundance, primary cell populations.
- Strategic Value: Reduces biological ambiguity and supports informed go/no-go decisions.
- Portfolio Impact: Enables risk-adjusted advancement of muscle biology programs.
Implementation Considerations
- Requires expertise in FACS, chromatin biology, and next-generation sequencing.
- Needs access to cell sorting, magnetic bead handling, and sequencing infrastructure.
- Demands rigorous cross-team standardization for sample preparation and data analysis.
- Adaptable to other rare cell types with protocol optimization.
- Limited by input cell number and antibody specificity for target proteins.
