White prepupae were collected and aged for 2 days. These were then dissected in saline by tearing them immediately posterior to the mouth hooks using paired forceps and everting them. Males and females (as identified by the clear gonadal tissue embedded in fat body of the A5 segment) were dissected separately, and preparations were combined in equal numbers for RNA processing. Fat body was isolated and frozen on dry ice.

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/).