Proper differentiation of sperm from germline stem cells, essential for production of the next generation, requires dramatic changes in gene expression that drive remodeling of almost all cellular components, from chromatin to organelles to cell shape itself. Here we provide a single nucleus and single cell RNA-seq resource covering all of spermatogenesis in Drosophila starting from in-depth analysis of adult testis single nucleus RNA-seq (snRNA-seq) data from the Fly Cell Atlas (FCA) study (Li et al., 2022). With over 44,000 nuclei and 6,000 cells analyzed, the data provide identification of rare cell types, mapping of intermediate steps in differentiation, and the potential to identify new factors impacting fertility or controlling differentiation of germline and supporting somatic cells. We justify assignment of key germline and somatic cell types using combinations of known markers, in situ hybridization, and analysis of extant protein traps. Comparison of single cell and single nucleus datasets proved particularly revealing of dynamic developmental transitions in germline differentiation. To complement the web-based portals for data analysis hosted by the FCA, we provide datasets compatible with commonly used software such as Seurat and Monocle. The foundation provided here will enable communities studying spermatogenesis to interrogate the datasets to identify candidate genes to test for function in vivo.

The “Testis, 10X, Relaxed” loom file (https://flycellatlas.org/) was imported into Seurat. A community of Drosophila researchers with interests in testis / spermatogenesis / fertility / and stem cell-niche biology adjusted and updated the previously reported cluster annotations (Li , 2022), and supported the assignments by analysis of markers using in situ hybridization and extant protein traps. Significant new metadata are provided in this Seurat object, including the new annotation assignments and trajectory inference for both the germline and somatic lineages. A monocle object is included seprately, representing trajectory inference analysis of the data set. The base files representing the scRNA-seq data are also included (barcodes, features and martix).

Examples:

Seurat 4.0

RStudio 2021.09.0

R 4.0.2