Nuclei Isolation Protocol for Single-Nucleus RNA Sequencing of Human Stem Cell-Derived Grafts

Single-nucleus RNA sequencing enables high-resolution transcriptomic profiling of brain tissue, facilitating detailed analysis of cell identity in models of neurodegeneration and repair. Here, we describe a protocol for isolating nuclei from long-term human stem cell–derived grafts in the rat brain, incorporating 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, offering a powerful approach to assess graft composition, integration, and neuronal identity in living brain.

For complete details on the use and execution of this protocol, please refer to Fiorenzano et al. ¹

Subject areas

Single nucleus RNA sequencing, stem cell biology, neuroscience, transplantation, cell replacement therapy

Graphical abstract

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Highlights

• Step-by-step vibratome sectioning of xenografted rat brain tissue.

• Precise dissection of human stem cell–derived grafts from host brain sections.

• Extraction of intact nuclei from fragile, grafted neurons.

• Isolation of single nuclei via fluorescence-activated nuclei sorting (FANS) for snRNA-seq sample preparation.