The development of the mouse central nervous system (CNS) involves coordinated execution of transcriptional and epigenetic programs. These programs have been extensively studied through single-cell technologies in a pursuit to characterize the underlying cell heterogeneity. However, histone modifications pose additional layers of both positive and negative regulation that defines cellular identity. Here we show that the Cut&Tag technology can be coupled with a droplet-based single cell library preparation platform to produce high quality chromatin modifications data at a single cell resolution in tens of thousands of cells. We apply single-cell Cut&Tag (scC&T) to probe histone modifications characteristic of active promoters (H3K4me3), active promoters and enhancers (H3K27ac), active gene bodies (H3K36me3) and inactive regions (H3K27me3) and generate scC&T profiles for almost 50,000 cells. scC&T profiles of each of these histone modifications were sufficient to determine cell identity and deconvolute at single cell level regulatory principles such as promoter bivalency, spreading of H3K4me3 and promoter-enhancer connectivity. Moreover, we used scC&T to investigate the single-cell chromatin occupancy of transcription factor Olig2 and the cohesin complex component Rad21. Our results indicate that analysis of histone modifications and transcription factor occupancy at a single cell resolution can provide unique insights of epigenomic landscapes in the CNS. We also provide an online resource that can be used to interactively explore the data at <https://castelobranco.shinyapps.io/BrainCutAndTag2020/>. ### Competing Interest Statement The authors have declared no competing interest.
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