Protocol for isolating nuclei from human stem cell-derived grafts for single-nucleus RNA sequencing.

Abstract

Single-nucleus RNA sequencing enables high-resolution transcriptomic profiling of brain tissue, facilitating analysis of cell identity in models of neurodegeneration and repair. Here, we present a protocol for isolating nuclei from long-term human stem cell-derived grafts in the rat brain. We describe steps for vibratome sectioning, graft dissection, nuclear extraction, and fluorescence-activated sorting. This workflow supports analysis of human neurons embedded within host tissue or sensitive to dissociation, enabling assessment of graft composition, integration, and neuronal identity in the living brain. For complete details on the use and execution of this protocol, please refer to Fiorenzano et al.¹.