Standardization of Single-Cell RNA-Sequencing Analysis Workflow to Study Drosophila Ovary

Developments in single-cell technology have considerably changed the way we study biology. Significant efforts have been made over the last few years to build comprehensive cell-type-specific transcriptomic atlases for a wide range of tissues in several model organisms in order to discover cell-type-specific markers and drivers of gene expression. One such tissue is the ovary of the fruit-fly Drosophila melanogaster, which is a popular model system with wide-ranging applications in the study of both development and disease. Three independent studies have recently produced comprehensive maps of cell-type-specific gene expression that describe both spatiotemporal regulation of the process of oogenesis and unique transcriptomic profiles of different cell types that constitute the ovary. In this chapter, we outlined the wet-lab protocol that was followed in our recent study for sample preparation and reanalyze the resultant dataset to discuss the benchmarks in data analysis, which are fundamental to comprehensive curation of the single-cell dataset representing the fly ovary.