Project Summary Single cell RNA-sequencing (scRNA-seq) has enabled researchers to investigate a wide array of biological processes and how tissue heterogeneity contributes to function. These technologies have led to the development of programs, such as the NIH Human Biomolecular Atlas Program, to understand how cells interact in an organism to drive its function. The development of spatial transcriptomic technologies has allowed researchers to investigate how cell-cell interactions affect cellular gene expression within a tissue. Current approaches generally rely in situ RNA based methodolgies or slide-based sequencing in combination with scRNA-seq (e.g. Slide-seq) to generate spatial maps of interacting cells. These technologies have helped demonstrate the power of combining the tissues structural data with transcriptional data to better understand cell behavior in a tissue and going forward improving the interface between these technologies will be key to uncovering how structure and cell transcription drives function. However, multiple barriers impede the widespread adoption of the current methods for performing spatial transcriptomics, including significant financial cost, time requirements, and a lack of sufficient expertise. Herein, we propose to develop a simple reagent compatible with current protocols for drop-based and pipette-based scRNA-seq technologies. This reagent will be able to generate spatial data in the normal course of scRNA-seq experiments without significantly increasing cost or time commitments. We propose to invent “antibody-oligonucleotide bridges” and build software capable of bioinformatically generating a network of cell-cell contacts across the tissue to recapitulate the spatial relationships of the cells in a tissue. In this proposal, we will carry out a proof-of- principal experiment for the AO bridge scRNA-seq approach for generating the spatial relationships between cells.