UC San Diego

DNA methylation at cytosines has long been associated with early development, maturation, and aging of human tissues. Traditionally, DNA methylation is associated with gene silencing. However, recent single cell multi-omic DNA methylation and RNA sequencing methods have shown that the role of DNA methylation on the expression of nearby genes could silence or activate them depending on the gene and cell type. The recent developments these assays have detected cell type specific DNA methylation and RNA coupling in stem cell rich and human brain tissues. This specificity underscores the need for future growth in DNA methylation and RNA co-sequencing technologies and analysis tools. Presently, about 100,000 single cell profiles are required to adequately map tissues. DNA methylation and RNA co-sequencing methods require the physical isolation of single cells in individual wells. There is no method that can assay 100,000 cells without utilizing extensive liquid handling systems. We address this challenge by developing a novel ultra-high throughput DNA methylation and RNA co-sequencing platform, sci-Gel, that utilizes three levels of combinatorial indexing to increase the throughput of existing technologies to 50,000-100,000 cells per experiment with just three 96 well plates. In this dissertation, we first push the boundaries of present combinatorial indexing techniques where the DNA and RNA of single cells are simultaneously extracted and immobilized within polyacrylamide gel beads that are used for indexing. This resulted in the development of a 2-level combinatorial indexing platform that could be used to co-sequence DNA copy-number variations, relevant in cancers, and RNA at the scale of thousands of cells. We then describe the adaptations made from existing bisulfite conversion chemistries to our gel beads to incorporate the DNA methylation feature. We then describe the development of a 3-level combinatorial indexing platform to increase the cell throughput of our technology to 50,000-100,000 cells per experiment. Finally, we discuss future efforts to utilize sci-Gel to create the first single cell DNA methylation and RNA co-sequencing map of peripheral blood mononuclear cells.