Preparation of Human and Rodent Spinal Cord Nuclei for Single-Nucleus Transcriptomic Analysis

The spinal cord is a complex and heterogeneous tissue that is composed of numerous neuronal and non-neuronal cell types. Single-cell RNA-seq has emerged as a powerful method to study heterogeneous tissues by allowing for the capture and analysis of individual cells. A key step in the analysis of solid tissues is the dissociation of the tissue into single cells. While many tissues can be readily dissociated by enzymatic or mechanical methods, highly interconnected tissues such as the spinal cord are especially difficult to dissociate, leading to reductions in cellular viability or large biases in the representation of cell types. Moreover, it has been shown that dissociation of live cells can induce gene expression changes that influence downstream analysis. Single-nucleus RNA-seq (snRNA-seq) offers an alternative method of studying the transcriptomes of individual cells that circumvents many of these issues. An additional benefit of snRNA-seq is that it can be performed on frozen tissues, thus opening the door to the study of biobanked pathological human tissues. We present here a straightforward protocol to isolate both murine and human spinal cord nuclei for transcriptomic analysis on multiple platforms, including 10× Genomics Chromium.