Integrated Analysis of Single-Cell Atlases and Droplet Microfluidics Based Single-Cell Sequencing Methods

Abstract

In this thesis, I present the development of a droplet microfluidics-based strategy for transposase-based DNA barcoding. While the method has general utility, the motivation for this thesis lies specifically in a high-throughput method for single-cell measurements of chromatin state. I establish a protocol in which Tn5 transposase is associated with sequencing adapters in the tagmentation reaction and the usually required pre-assembly step can be skipped. The compatibility of this approach with droplet microfluidics and sequencing adapters assembled on hydrogels is demonstrated. Together, this provides the basis for transposase based barcoding in droplet microfluidics. Furthermore, this thesis explores integrated analyses across human cell atlases in the context of the COVID-19 pandemic: ACE2 and accessory proteases (TMPRSS2, CTSL) are needed for SARS-CoV-2 cellular entry, and their expression may shed light on viral tropism and impact across the body. This work assesses the cell type-specific expression of ACE2, TMPRSS2, and CTSL across 107 single-cell RNA-Seq studies from different tissues. ACE2, TMPRSS2, and CTSL are co-expressed in specific subsets of respiratory epithelial cells in the nasal passages, airways, and alveoli, and in cells from other organs associated with COVID-19 transmission or pathology. We performed a meta-analysis of 31 lung scRNA-seq studies with 1,320,896 cells from 377 nasal, airway, and lung parenchyma samples from 228 individuals. This revealed cell type specific associations of age, sex, and smoking with expression levels of ACE2, TMPRSS2, and CTSL. Expression of entry factors increased with age and in males, including in airway secretory cells and alveolar AT2 cells. Expression programs shared by ACE2+TMPRSS2+ cells in nasal, lung and gut tissues included genes that may mediate viral entry, key immune functions and epithelial-macrophage cross-talk, such as genes involved in the IL6, IL1, TNF and complement pathways. Cell type-specific expression patterns may contribute to COVID-19 pathogenesis , and our work highlights putative molecular pathways for therapeutic intervention.