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Abstract

Single-cell analysis has become an increasingly important tool in cell biology. One of the most popular analysis methods is single-cell ribonucleic acid sequencing (scRNA-seq), which enables quantification of gene expression in single cells. This type of information has led to many important discoveries, from existence of new cell types to tumor heterogeneity. However, it is proteins, not RNAs, that are the main effector molecules in biological processes. Cells recognize environmental signals using protein receptors on the cell surface. The environmental signals are then transmitted through protein interactions inside the cells as part of a signaling cascade. Finally, the appropriate genes are transcribed, allowing the cells to respond to the environmental signals. These protein activities cannot be inferred from gene expression level alone, because many of them occur prior to transcription. In this dissertation, we introduce Proximity sequencing (Prox-seq), a novel single-cell multiomic method that bridges the gap between gene expression and protein-protein interactions. Prox-seq combines proximity ligation assay (PLA) with scRNA-seq to quantify gene expression level, protein abundance, and most importantly, protein complexes in the same single cell. First, we develop Prox-seq for surface receptors, and show that we can indeed obtain these three types of information from the same single cells. By applying Prox-seq to peripheral blood mononuclear cells (PBMCs), we find a putative interaction between CD8 and CD9 receptors on CD8 T cells. Then, we develop a high-throughput, droplet-based scRNA-seq for fixed and permeablized cells, called FD-seq. FD-seq can serve as a platform to extend Prox-seq to intracellular proteins. Using FD-seq, we identify host genes that are associated with herpesvirus reactivation, and show that following exposure to coronavirus, only a minority of the cells express a high level of viral genes. Finally, we develop computational frameworks for simulating Prox-seq data, and for prediction of protein complexes in single cells from Prox-seq data.

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