SRX18551197 GSM6807401_r1 SRP412210 PRJNA910535 SRS16017429 GSM6807401 GSM6807401 RNA-Seq TRANSCRIPTOMIC SINGLE CELL cDNA The testis dissociation protocol was adapted from Witt et al., 2019. Fresh maceration buffer (10mL Trypsin LE (Gibco) with 20 mg collagenase (Gibco)) was prepared on the day of the dissection. Testes were hand dissected from 1-5 day old male flies in 1x phosphate-buffered saline (PBS), separated from seminal vesicles and transferred immediately into tubes filled with cold PBS, on ice. Testes were kept in PBS for a maximum of 30 minutes. Samples were centrifuged at 135 rcf and the PBS removed and replaced with 400 μL marceration buffer. Testes were incubated in maceration buffer for 30 minutes with gentle vortexing every 10 minutes at room temperature. Following incubation, samples were pipetted up and down for 15 minutes until all visible chunks were gone and the sample was in approximately a single-cell suspension. Sample was filtered through a 35 μm filter into a polystyrene tube, then transferred into a microcentrifuge tube. After the sample was centrifuged at 135 rcf for 7 minutes, the supernatant was removed and the pellet resuspended in 1mL calcium- and magnesium-free Hanks' Balanced Salt Solution (HBSS). The sample was spun a final time at 137 rcf for 7 minutes. All but 50 μL of the HBSS supernatant was removed, and the cell pellet was resuspended in the remaining 50 μL. Cell viability and density was then assayed on a hemocytometer using DIC imaging and Trypan Blue stain. Cells were processed using the 10x Genomics Chromium Controller and Chromium Single Cell Library and Gel Bead Kit following standard manufacturer's protocol. Amplified cDNA libraries were quantified by bioanalyzer and size selected using AMPure beads. Samples were sequenced on a NovaSeq SP. Illumina NovaSeq 6000