show Abstracthide AbstractPurpose: After identifying a difference in the response to sleep deprivation between the morning and evening, we assayed gene expression in a key homeostatic circuit in the Drosophila brain: the R5 ellipsoid body neurons. We sought to identify changes in gene expression in this circuit that are critical for the behavioral output. Methods: Flies expressing GFP via a restrictive R5 driver were entrained to a 12:12 light:dark paradigm at 25C. Mated females were then loaded into individual tubes with sucrose food and placed into behavior boards from Trinkinetics. Control flies were not subjected to any sleep deprivation and were collected in either the morning or evening. Experimental flies were subjected to 2.5 hrs of mechanical sleep deprivation (SD) via vortexing just before either the morning or evening. Following SD (or at the appropriate control timepoint), 40-50 brains were dissected out in dissecting saline with TTX, AP5, and DNQX to inhibit sodium channels, NMDA receptors, and AMPA receptors respectively. The brains were transferred to SM Active medium with channel blockers after dissection, then washed with dissecting saline before being digested with papain. Digestion was stopped after 30 minutes by the addition of SM Active medium with channel blockers. The brains were then triturated by manual pipetting to produce a single cell suspension. GFP+ cells were immediately sorted by fluorescence activated cell sorting into a single tube. 350-500 cells were collected per experiment. In total, we collected 12 samples, three replicates for each timepoint and condition. Cells were lysed in extraction buffer from the Arcturus PicoPure kit and stored at -80C until ready to process. RNA was extracted from each set of cells using the PicoPure kit, and then reverse transcribed into cDNA using a T7/polydT primer. One round of in vitro transcription using the MEGAScript kit was performed to amplify the RNA. Following IVT, library prep was performed using the TruSeq Stranded mRNA Library Prep Kit. We generated 30 million reads per sample on a NovaSeq. Analysis was performed using kallisto and sleuth. Overall design: mRNA from R5 ellipsoid body neurons of Drosophila in the morning and evening +/- SD