White prepupae were collected and aged for 2 days. These were dissected in saline by removing the posterior at the A7 abdominal segment using forceps. The anterior of the pupa was removed from the cuticle, the CNS and ring gland were isolated, and frozen on dry ice.

Total RNA was isolated using Trizol, then Qiagen RNeasy spin columns. mRNA was isolated using Dynal oligo(dT) beads, then fragmented using divalent cations under elevated temperature, followed by sequential ligation of RNA linkers to the 5’ and 3’ ends. Next, reverse transcription was performed using a primer complementary to the 3’ linker and PCR was performed using primers complementary to both linkers. ~ 300 bp fragments were isolated from an agarose gel and gel-purified again.