Single nucleus RNA-sequencing defines unexpected diversity of cholinergic neuron types in the adult mouse spinal cord

We developed a targeted single nucleus RNA sequencing approach to enrich and transcriptomically characterize cholinergic neurons of the adult mouse spinal cord. Our data expose markers for known classes of cholinergic neurons and their extremely rich diversity. Visceral/preganglionic motor neurons could be divided into more than a dozen transcriptomic classes with anatomically restricted localization along the spinal cord. The complexity of the skeletal motor neurons was also reflected in our analysis with alpha, gamma, and a third subtype clearly distinguished. We further identified previously unrecognized subtypes of cholinergic interneurons. Overall design: Cholinergic nuclei were enriched by FACS sorting of GFP+ nuclei from nuclear reporter mice and barcoded using 10X Genomics Chromium Single Cell Processor. Samples were run on an Illumina HiSeq2500.