Isolation of stage-specific germ cells using FACS in Drosophila germarium.

Abstract

Drosophila melanogaster oogenesis is a versatile model system to address many fundamental questions of cell and developmental biology, such as stem cell biology, mitosis, meiosis or cell polarity. Many mutagenesis and powerful genetic tools have contributed massively to identify and dissect in vivo gene functions in a stage and tissue specific manner. However, the small number of germ cells during the early steps of oogenesis have hampered a systematic description of RNA and protein contents at each stage. We describe here a protocol for isolating and comparing two small subpopulations of cells in the ovary for the purpose of RNA sequence profiling. The method is based on fluorescence-activated cell sorting (FACS) of GFP- and RFP-labeled proteins that are expressed in distinct and mostly non-overlapping regions of the germline. We used a transgene expressing a GFP-tagged Bam protein driven by its own promoter, labeling specifically the mitotic region of the germarium. We also took advantage of the short-lived Wicked protein tagged with RFP and expressed under the nanos promoter to label the meiotic region. We generated flies expressing both markers and were able to sort enough cells from each region to extract total RNAs and small RNAs. Total RNA or small RNA extracted from sorted cells were then used to generate deep-sequencing libraries that show specificity toward each compartment. This method of isolating a very small number of cells and the data generated from comparing distinct cell populations within the germline should further our understanding of these conserved steps of oogenesis.