Anaesthetized adult files were pinned down and T3 legs were removed to expose internal tissues. The digestive tract was dissected 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.