Mass spectrometry analysis of metabolites from sorted somatic cyst cells (files labelled Soma) and germ cells (files labelled Germ) from dissected Drosophila testes. Files are separated into positively-charged (Soma1 to 6 and Germ1 to 6) and negatively-charged metabolites (Soma1-2 to Soma6-2 and Germ1-2 to Germ 6-2).Cell dissociation, sorting and metabolite extraction:Drosophila testes were dissected in Schneider’s medium and then separation buffer containing Schneider’s medium, collagenase, trypsin and EDTA was added. Samples were vigorously agitated for 15-30 min. The resulting cell suspension was then filtered using a cell strainer. GFP+ somatic cells and GFP-germ cells were sorted using a BD FACSAria Fusion Cell Sorter. Cells were spun down at 1550 RPM for 10 minutes at 4°C, resuspended in 1:3:1 chloroform/methanol/water for lysis and incubated for 1h at 4°C with rocking. Lysates were spun down at 13,000 RPM for 3 minutes at 4°C and supernatants were extracted and stored at -80°C. 6 replicates were obtained. Mass spectrometry:Mass spectrometry analysis of the samples was carried out at the Polyomics Facility at the University of Glasgow. Hydrophilic interaction liquid chromatography (HILIC) was carried out on a Dionex UltiMate 3000 RSLC system (Thermo Fisher Scientific) using a ZIC-pHILIC column (150 mm × 4.6 mm, 5 μm column, Merck Sequant). Samples were eluted with a linear gradient of 20 mM ammonium carbonate in water and acetonitrile. For the MS analysis, a Thermo Orbitrap QExactive (Thermo Fisher Scientific) in polarity switching mode was used. To confirm the identity of metabolites, standards were run alongside the samples to match signals based on accurate mass and retention time. Signals that did not match authentic standards were annotated based on accurate mass. As an output, 6 .mzXML files for positively-charged metabolites obtained from somatic and germ cells were obtained (labelled as Soma1 to 6 and Germ1 to 6), as well as 6 files for negatively-charged metabolites isolated from the same cell populations (labelled as Soma1-2 to Soma6-2 and Germ1-2 to Germ6-2).
