Defining Selective Neuronal Resilience and Identifying Targets for Neuroprotection and Axon Regeneration Using Single-Cell RNA Sequencing: Experimental Approaches.

A prevalent feature among neurodegenerative conditions, including axonal injury, is that certain neuronal types are disproportionately affected, while others are more resilient. Identifying molecular features that separate resilient from susceptible populations could reveal potential targets for neuroprotection and axon regeneration. A powerful approach to resolve molecular differences across cell types is single-cell RNA-sequencing (scRNA-seq). scRNA-seq is a robustly scalable approach that enables the parallel sampling of gene expression across many individual cells. Here we present a systematic framework to apply scRNA-seq to track neuronal survival and gene expression changes following axonal injury. Our methods utilize the mouse retina because it is an experimentally accessible central nervous system tissue and its cell types have been comprehensively characterized by scRNA-seq. This chapter will focus on preparing retinal ganglion cells (RGCs) for scRNA-seq and pre-processing of sequencing results.