A cellular cross-species RNA-seq atlas captures the transcriptional dynamics of myogenesis

Skeletal muscle formation and regeneration is a tightly regulated process involving extensive transcriptional reprogramming as proliferating myoblasts fuse into mature myotubes. However, a comparative comprehensive analysis of the transcriptional landscape of humans and mouse myogenesis is missing. Here, we present a high-quality RNA-sequencing dataset profiling this transition in both mouse (C2C12) and human (LHCN-M2) myogenic cells. Samples were collected from proliferating myoblasts, the early differentiation phase, and from mature myocytes using identical protocols, ensuring stringent comparability. This unified dataset captures the major transcriptional shifts occurring in myoblasts, marking the onset of differentiation. Quality metrics, including PCA, read distribution, and clustering, confirmed high internal consistency across samples and species. Comparative analyses revealed shared global features of myogenesis but also distinct regulatory trajectories. Human differentiation showed early upregulation followed by suppression of metabolic and stress-related pathways, while structural and ECM-associated programs remained persistently elevated. In contrast, mouse C2C12 cells displayed early inflammatory activation and later enrichment of metabolic and contractile pathways typical of mature myotubes. Ortholog-based integration demonstrated decreasing cross-species correlation over time, indicating progressive reinforcement of species-specific differentiation programs. We additionally compared this bulk RNA-seq with tissue-derived myotubes from single-cell muscle datasets to determine which transcriptional programs are conserved in the in vitro models or which emerge only in vivo. This analysis delineates the conserved and context-specific features of myogenesis, identifying pathways that reflect culture-specific artifacts in both human and mouse muscle cells.