Flies were collected at circadian time ZT10. 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.
RNA sequences were mapped to the reference genome using TopHat. Potential A-to-I editing sites were only considered if they were observed in at least ten of 12 replicates and no G residues were observed at the same position in parallel DNA-Sequencing of the same strain (39X coverage).