Novel Multiomics Method of RNA and Nuclear Protein Characterization in Single Cells
- Author(s): Jacobsen, Daniel Eric
- Advisor(s): Zhang, Kun
- et al.
Characterizing single cells has become an important field in understanding human structure and function, both in healthy and diseased individuals. Recently, this characterization has included multi-omics methods, which use complementary data sets to better understand cellular function from several angles. RNA-seq is a staple for characterizing cell phenotypes, but fails to cover the entire picture of the cells phenotype. Protein expression has long been considered as a primary driver in characterizing cells, but inefficiencies in capturing meaningful protein data has long been a problem. Recently, antibodies conjugated to oligonucleotides have helped address these inefficiencies, bringing the scale of single-cell proteomics assays more on par with single-cell RNA assays. However, these assays have characterized only cell-surface marker proteins, and have not yet shown an ability to capture proteins with more meaningful intracellular data. Of deeper interest are intranuclear transcription factors, which are directly related to RNA expression patterns. This dissertation describes strategies of single-cell protein and RNA capture, including design of probe oligonucleotides and antibody conjugation methods. It also describes use of antibody fragments and the small molecule pitstop 2 as a method of intranuclear protein assaying. Finally, the dissertation describes dual RNA-protein characterization of cell cycle in thousands of cultured cells, and additional conclusions drawn from this combined data.