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Summary: Single-nucleus RNA sequencing allows the profiling of gene expression in isolated nuclei. Here, we describe a step-by-step protocol optimized for adult mouse skeletal muscles. This protocol provides two main advantages compared to the widely used single-cell protocol. First, it allows us to sequence the myonuclei of the multinucleated myofibers. Second, it circumvents the cell-dissociation-induced transcriptional modifications.For complete details on the use and execution of this protocol, please refer to Dos Santos et al. (2020) and Machado, Geara et al. (2021).