Preparation of Drosophila Larval Blood Cells for Single-cell RNA Sequencing

Recent advances in single-cell RNA-sequencing (scRNA-seq) technologies provide unprecedented opportunities to identify new cell types and characterize cell states. One of the most important requirements for performing scRNA-seq is to obtain high-quality single cells in suspension. Recently, we used this approach to characterize Drosophila blood cells (hemocytes). Here, we provide a detailed protocol for obtaining single hemocytes in suspension, which can be used for microfluidics-based scRNA-seq platforms. This protocol involves the simple bleeding of third instar larvae and the subsequent purification of the hemolymph using either Optiprep-based gradient centrifugation or traditional centrifugation methods to obtain single hemocytes of high quality for scRNA-seq. Importantly, this method for single-hemocyte preparation is straightforward and reproducible, with negligible issues associated with cell viability as the entire procedure involves no enzymatic dissociation. Graphic abstract: Workflow for the preparation of Drosophila larval blood cells in suspension. Hemocytes (blood cells) of the sessile and circulatory compartments of larvae are derived by simple bleeding and purification using gradient centrifugation. Blood cells are counted and subsequently encapsulated by microfluidics-based scRNA-seq platforms. Blood cells represented in the schematic are derived from third instar larvae of the genotype Hemolectin-GAL4.Delta, UAS-2xEGFP (BDSC stock #30140).