High-throughput droplet-based single-cell small RNA sequencing technology

Droplet-based single-cell RNA-sequencing (scRNA-seq) technologies have penetrated almost all branches of life sciences and have significantly advanced our understanding of cellular processes and organism development. However, despite their astonishing impact, most of the scRNA-seq technologies reported to date rely on poly(A) tail capture and thus are mainly restricted to the protein-coding RNAs, while neglecting a substantial proportion of the transcriptome, including small non-coding RNAs. As a result, very little is known about the non-coding RNA expression and function in individual cells, and especially their role in the establishment of cellular phenotypic diversity. Small RNAs contain a variety of classes, of which miRNAs are the most common and these act as regulatory molecules by suppressing translation of mRNAs. In addition, loss-of-function studies of miRNAs uncovered their involvement in development of nearly all tissues, including hematopoiesis. However, most studies exploring miRNA dynamics reported to date relied on bulk cell assays, thus disregarding the individual cell types and their heterogeneity. In the scope of this proposal, we aim to develop a high-throughput droplet-based single-cell small RNA-seq (droplet-small-seq) for simultaneous miRNA and mRNA capture and sequencing. We will apply this newly developed technique to investigate the regulatory roles of miRNAs in cell fate decision during hematopoietic development at a single-cell level.