Single Cell sequencing of mononuclear cells from regenerating skeletal muscle

Skeletal muscle stem cells, or satellite cells (SCs), are essential to regenerate and maintain muscle. Quiescent SCs reside in an asymmetric niche between the basal lamina and myofiber membrane. To repair muscle, SCs activate, proliferate, and differentiate, fusing to repair myofibers or reacquiring quiescence to replenish the SC niche. Little is known about when SCs reacquire quiescence during regeneration or the cellular processes that direct SC fate decisions and progression through myogenesis. Single cell sequencing of myogenic cells in regenerating muscle identifies SCs reacquiring quiescence and reveals that non-cell autonomous signaling networks influence SC fate decisions during regeneration. Single cell RNA-sequencing of regenerating skeletal muscle reveals that RBP expression, including numerous neuromuscular disease-associated RBPs, is temporally regulated in skeletal muscle stem cells and correlates to stages of myogenic differentiation. By combining machine learning with RBP engagement scoring, we discover that the neuromuscular disease associated RBP Hnrnpa2b1 is a differentiation-specifying regulator of myogenesis controlling myogenic cell fate transitions during terminal differentiation. Mononuclear cells were isolated from mouse tibialis anterior muscle at 4 and 7 days following injury with Barium Chloride. The muscle was mechanically dissociated and digested with collagenase prior to affinity based removal of debris and dead cells. The isolated cells were barcoded for seqeuncing using the Chromium Next GEM Single Cell 3' Reagent kit v2.