Flies were collected at circadian time ZT18. Whole flies were frozen and adult heads were isolated using brass sieves. Tissue was homogenized on ice, filtered to remove debris, and centrifuged on a sucrose cushion to isolate nuclei.
Flies were entrained under 12hr:12hr light:dark cycles for 3-4 days.
Total RNA was extracted from isolated nuclei using Trizol. Replicate 1 was depleted of ribosomal RNA, replicate 2 was not. For both replicates, mRNA was depleted by two rounds of poly(A) RNA depletion. Sequencing library was constructed per standard Illumina protocol.
