Methods of muscle atrophy analysis at the single-nucleus level

Muscle atrophy and functional decline are shared manifestations of aging and neuromuscular pathologies. Deciphering the molecular mechanisms underlying muscle decline in these conditions has been slow, partly due to the difficulties of molecularly characterizing muscle fibers (the most abundant cell type in skeletal muscle). In contrast to single-cell RNA sequencing (scRNA-seq), which cannot resolve multinucleated fibers, single-nucleus RNA sequencing (snRNA-seq) enables gene expression profiling from isolated nuclei of hard-to-dissociate solid tissues, providing a key advantage for studying skeletal muscle. This paper presents a detailed protocol for proper tissue dissociation and nuclei isolation from skeletal muscle, optimized for downstream transcriptomic analysis. Additionally, we outline a basic bioinformatics pipeline applicable to snRNA-seq data from skeletal muscle, focusing on transcriptomic comparisons between homeostatic and atrophic muscle states.