Odor-cue-induced ethanol memory was performed as published previously (Kaun et al. 2011; Nunez et al. 2018; Petruccelli et al. 2018). One minor difference for the RNA-seq data were the use of 50 flies per 14 ml training vial for the isolation of nuclei tagged in specific cell types (INTACT) experiment, whereas memory experiments were performed with 30 flies per vial. Humidified ethanol vapor (90:60 EtOH:air) for 10 min causes a moderate 13.8 ± 3 mM (0.01 g/dl) internal body ethanol concentration. Ethanol, odors (1:36 odorant:mineral oil with ethyl acetate or isoamyl alcohol), or both were delivered to flies in perforated vials for 10 min, followed by a 50 or 60 min rest before second and third trainings were performed (Figure 1A). Vapor treatments were delivered to flies on 1% agar and supplemented with yeast pellets overnight before being tested or killed the next day.
