Epigenomic Diversity of Cortical Projection Neurons in the Mouse Brain

Neuronal cell types are classically defined by their molecular properties, anatomy, and functions. While recent advances in single-cell genomics have led to high-resolution molecular characterization of cell type diversity in the brain, neuronal cell types are often studied out of the context of their anatomical properties. To better understand the relationship between molecular and anatomical features defining cortical neurons, we developed Epi-Retro-Seq, a method that combined retrograde labeling with single-nucleus DNA methylation sequencing to link epigenomic properties of cell types to neuronal projections. We profiled 16,971 single neocortical neurons from 63 cortico-cortical and cortico-subcortical long-distance projections. Our results revealed unique epigenetic signatures of projection neurons that correspond to their laminar and regional location and projection patterns. The methylomes of 16,971 mouse neocortical neurons from 63 cortico-cortical (CC) and cortico-subcortical long-distance projections were analyzed using Epi-Retro-Seq, a method that combined retrograde tracing and single nucleus methylome sequencing. In Epi-Retro-Seq, the retrograde viral tracer rAAV2-retro-Cre is injected in the target region in an INTACT mouse, turning on Cre-dependent nuclear-GFP expression in neurons that project to the injected target, throughout the mouse brain. The brain is then sectioned into eighteen 600-micron coronal slices, and the brain regions of interest are mannually dissected from each slice. Nuclei are sampled from at least 4 mice (2 male and 2 female) for each projection target (except AI→pons - 2 male mice only). Nuclei from each of the dissected source regions are prepared, from which GFP+/NeuN+ nuclei (the GFP-labeled projection neurons) are isolated as single nuclei using fluorescence activated nuclei sorting (FANS) and assayed using snmC-Seq2 to profile their genome-wide DNA methylation signatures. We assayed approximately 384 nuclei from each projection (except the MOp→SSp projection from which 768 nuclei were assayed). Low-quality nuclei were removed.