Anaesthetized adults were placed in a 15ml conical tube, flash frozen in liquid nitrogen, then shaken vigorously for 10 seconds. Broken flies were placed on a Petri dish on dry ice and frozen severed heads were removed with forceps to a fresh tube. Flies were processed in groups of 100 animals per dissection. Typically, heads were missing the antennal and maxillary organs, while the mouth-parts were retained.

Total RNA was primed for first strand cDNA synthesis using a random N15 primer in the presence of trehalose and sorbitol. The RNA/cDNA hybrid was purified, then capped RNA was biotinylated and purified using streptavidin beads. The first strand cDNA was ligated to another primer at the 5' end for second strand cDNA synthesis. The resulting double-stranded cDNA was cleaved using EcoP15I and a second linker was ligated to the 3' end of the ds-cDNA products. 5'-CAGE tags were purified by streptavidin then amplified by PCR.

The 9-nt barcode linker sequence was removed, and the 27-nt CAGE reads representing capped 5’ transcript ends were aligned to the D. melanogaster genome using StatMap (http://www.statmap-bio.org/).