Single-cell analysis has become a powerful approach for the molecular characterization of complex tissues. Methods for quantifying gene expression and DNA accessibility of single cells are now well-established, but analysis of chromatin regions with specific histone modifications has been technically challenging. Here, we adapt the recently published CUT&Tag method to scalable single-cell platforms to profile chromatin landscapes in single cells (scCUT&Tag) from complex tissues. We focus on profiling Polycomb Group (PcG) silenced regions marked by H3K27 trimethylation (H3K27me3) in single cells as an orthogonal approach to chromatin accessibility for identifying cell states. We show that scCUT&Tag profiling of H3K27me3 distinguishes cell types in human blood and allows the generation of cell-type-specific PcG landscapes from heterogeneous tissues. Furthermore, we use scCUT&Tag to profile H3K27me3 in a brain tumor patient before and after treatment, identifying cell types in the tumor microenvironment and heterogeneity in PcG activity in the primary sample and after treatment.

In the neocortex, subcerebral axonal projections originate largely from layer 5 (L5) extratelencephalic-projecting (ET) neurons. The highly distinctive morpho-electric properties of these neurons have mainly been described in rodents, where ET neurons can be labeled by retrograde tracers or transgenic lines. Similar labeling strategies are not possible in the human neocortex, rendering the translational relevance of findings in rodents unclear. We leveraged the recent discovery of a transcriptomically-defined L5 ET neuron type to study the properties of human L5 ET neurons in neocortical brain slices derived from neurosurgeries. Patch-seq recordings, where transcriptome, physiology and morphology are assayed from the same cell, revealed many conserved morpho-electric properties of human and rodent L5 ET neurons. Divergent properties were also apparent but were often smaller than differences between cell types within these two species. These data suggest a conserved function of L5 ET neurons in the neocortical hierarchy, but also highlight marked phenotypic divergence possibly related to functional specialization of human neocortex.