Low-Input RNA-Sequencing of In Situ Fixed Adult Murine Muscle Stem Cells

Muscle stem cells (MuSCs) play a crucial role in skeletal muscle regeneration. Under homeostatic conditions, the majority of MuSCs remain quiescent, a reversible, nonproliferative state with minimal cell activity. Upon muscle injury, MuSCs rapidly activate, proliferate, differentiate, and form nascent muscle fibers. Notably, the tissue dissociation process in conventional cell isolation protocols disrupts the stem cell niche environment, exposes the MuSCs to extracellular stimuli, and swiftly changes the transcriptomic profiles of MuSCs, making it challenging to study the regulation of MuSC quiescence. In vivo fixation allows the preservation of MuSC quiescence for further analysis and profiling. This chapter will focus on the detailed downstream RNA-seq library preparation workflow for fixed MuSCs, including RNA purification and decrosslinking, cDNA synthesis, and tagmentation-based library construction.