Joint profiling of multiplex chromatin interactions, gene expression, and RNA-chromatin associations in single cells of the human brain

The dynamically organized chromatin complexes often involve multiplex chromatin interactions and sometimes chromatin-associated RNA. Here, we introduce the Multi-Nucleic Acid Interaction Mapping in Single Cell (MUSIC) technique to enable simultaneous profiling of multiplex chromatin interactions, gene expression, and RNA-chromatin associations within a single nucleus. We applied MUSIC to profile >9,000 single nuclei in the human frontal cortex. MUSIC-derived single-nucleus transcriptomes provide a comprehensive categorization of cortical cell types, subtypes, and cellular states. The genomic sequences of highly expressed genes frequently co-complex with their flanking genomic regions, forming Gene-Expression-Associated Stripes (GEAS), which exemplify the intricate coordination between transcription and chromatin architecture at the singlecell level. Additionally, we observed significant heterogeneity among female cortical cells in the association between the XIST long non-coding RNA (lncRNA) and the X chromosome (XIST-chrX association, quantified as XAL). Cells with high XAL demonstrated a greater difference in spatial organization between the XIST-associated (Xi) and non-associated (Xa) X chromosomes compared to XAL-low cells. Notably, excitatory neurons displayed enrichment in XAL-high cells and exhibited a more pronounced disparity in spatial organization between Xi and Xa compared to other cell types. The MUSIC technique offers a powerful tool for future investigations into chromatin architecture and transcription at a cellular resolution within complex tissues.