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Methods of muscle atrophy analysis at the single-nucleus level.

Mercedes Grima-Terrén1, Megan Rommelfanger2, Silvia Campanario1

  • 1Altos Labs, San Diego Institute of Science, San Diego, CA 92121, USA; Department of Medicine and Life Sciences, Universitat Pompeu Fabra (UPF), Barcelona 08003, Spain; Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), Madrid 28029, Spain.

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This study introduces a single-nucleus RNA sequencing (snRNA-seq) protocol for skeletal muscle, enabling detailed molecular analysis of muscle atrophy and aging. The method overcomes limitations of single-cell RNA sequencing for studying these complex neuromuscular conditions.

Keywords:
AtrophySingle-nucleusSkeletal muscle

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Area of Science:

  • Molecular Biology
  • Genomics
  • Skeletal Muscle Physiology

Background:

  • Muscle atrophy and functional decline are common in aging and neuromuscular diseases.
  • Studying skeletal muscle molecular mechanisms is challenging due to its multinucleated fiber structure.
  • Existing methods like single-cell RNA sequencing (scRNA-seq) cannot resolve multinucleated muscle fibers.

Purpose of the Study:

  • To present a detailed protocol for skeletal muscle tissue dissociation and nuclei isolation for transcriptomic analysis.
  • To establish a bioinformatics pipeline for analyzing single-nucleus RNA sequencing (snRNA-seq) data from skeletal muscle.
  • To facilitate transcriptomic comparisons between homeostatic and atrophic muscle states.

Main Methods:

  • Optimized tissue dissociation techniques for skeletal muscle.
  • Nuclei isolation procedures suitable for downstream transcriptomic analysis.
  • Development of a basic bioinformatics pipeline for snRNA-seq data analysis.

Main Results:

  • A robust protocol for preparing skeletal muscle nuclei for snRNA-seq.
  • A functional bioinformatics pipeline for analyzing skeletal muscle transcriptomic data.
  • Demonstrated feasibility of comparing gene expression in homeostatic versus atrophic muscle.

Conclusions:

  • Single-nucleus RNA sequencing (snRNA-seq) is a powerful approach for studying skeletal muscle molecular biology.
  • The provided protocol and pipeline enable deeper insights into muscle atrophy and aging.
  • This methodology can advance understanding of neuromuscular pathologies.