Protocol to analyze changes in hippocampal neural stem cell quiescence from single-cell RNA sequencing data

Here, we present a protocol to isolate mouse hippocampal neural stem cells from single-cell RNA sequencing datasets using a computational approach. We describe steps to analyze whether a perturbation affects quiescence depth. Specifically, we detail procedures for sequential reclustering, pseudotime trajectory analysis, and statistical testing of distribution shifts from deep to shallow quiescence. This protocol overcomes challenges in distinguishing quiescent neural stem cells from astrocytes and proliferating neural stem cells from progenitor cells. For complete details on the use and execution of this protocol, please refer to Rigo et al.¹.