Individual virgin female flies were housed separately in 65 mm × 5 mm glass tubes (Trikinetics, Waltham,
MA) containing 5% agarose with 2% sucrose. Parafilm with pinholes poked in it was used to cover
the open end of each tube. 2- to 5-day old flies were entrained under standard light–dark conditions,
with a 12 hr light phase and followed by 12 hr dark phase for 3–4 days prior to collection of data for
sleep analysis. Locomotor activity was collected with DAM System monitors (Trikinetics) in 1 min bins
as previously described (Agosto et al., 2008).