Background

Gene expression is dynamically regulated in dorsal root ganglion (DRG) cells after peripheral nerve injury, a process important for axonal regeneration and the neuronal hyperexcitability that contributes to neuropathic pain. Previous studies have analyzed gene expression and chromatin structure from bulk DRG tissues (Perkins et al., 2014; Chandran et al., 2016), but their interpretation is complicated by the cellular heterogeneity of the DRG.

Single-cell/single-nucleus genomics has become an important tool for studying molecular features within distinct peripheral sensory neuronal subtypes (Usoskin et al., 2015; Nguyen et al., 2017 and 2019; Zeisel et al., 2018; Sharma et al., 2020). While single-cell transcriptional profiling from sensory ganglia appears to provide greater transcriptional diversity than single-nucleus transcriptional profiling, a key advantage of analyzing nuclei is that it can be applied to both fresh and archived frozen specimens. This offers flexibility in sample preparation and greater access to human samples. Nuclei isolation is also associated with induction of immediate early genes contrary to whole-cell isolation because it is faster, avoids cytoplasmic signaling cascades, and is conducted entirely at 4°C. These features make single-nucleus analyses particularly attractive for studying changes that occur rapidly after a stimulus.

Here, we describe a density gradient centrifugation protocol for purifying nuclei from frozen mouse DRG samples, which is compatible with droplet-based single-nucleus genomic analyses (Renthal et al., 2020). This protocol, which is based on previous protocols for extracting mouse and human CNS nuclei (Mo et al., 2015; Renthal et al., 2018), yields a greater fraction of neuronal nuclei (40-50% neurons as measured by single-nucleus RNA sequencing) as compared with commonly used non-gradient dissociation methods (5-10% neurons) (Habib et al., 2017; Avraham et al., 2020; Slyper et al., 2020), while still sampling a broad range of non-neuronal DRG cell types.