In situ Single-Cell Multi-Omic and Morphological Profiling in Mammalian Brains

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PROJECT SUMMARY Single-cell technologies have revolutionized the characterization of mammalian brains allowing unbiased census of cell types and their transcriptomic and epigenomics signatures. However, the mapping of molecular signatures onto three-dimensional brain structures remains highly challenging since most single-cell methods can only analyze disassociated cells or nuclei. We propose to develop photonic-indexing sequencing (pi-seq) strategies for in situ spatial barcoding with single-cell resolution. The proposed pi-seq strategy writes high complexity molecular barcodes into the tissue using sequential ligation of DNA indices with the ligation reaction controlled by high-resolution patterned illumination. Chromatin accessibility and cytosine modifications are well-established epigenomics marks playing critical roles in transcription regulation in normal and disease tissues. We will integrate pi-seq with existing single-cell epigenomics techniques to develop methods for the spatial profiling of chromatin accessibility (pi-ATAC-seq) and methylcytosine (pi-mC-seq). We will further develop an in situ method pi-mCAT-seq to simultaneously profile RNA, methylcytosine, and chromatin accessibility at a single-cell resolution based upon our single-nucleus multi-omics method snmC2T-seq. The spatial specificity and data quality of pi-seq methods will be systematically evaluated using single-cell epigenomic datasets generated by BICCN. To connect molecular profiles with other defining properties of brain cell types such as morphology and synaptic connectivity, we will develop methods to integrate pi-seq with MORF (Mosaicism with Repeat Frameshift), a sparse and genetic labeling approach of neurons and glia to illuminate their complete morphologies (dendrites, axons, synapses). The proposed pi-seq methods will provide the tools to construct spatially resolved epigenomic atlas of mammalian brains and advance the study of gene regulation in brain development, function, and disease at the resolution of single cells.