Newly eclosed flies were kept at 25oC for 84hr. Aged, mated flies were transferred to empty glass vials and the temperature was decreased to 0oC at a rate of 0.2 degrees per minute; the flies were then held at 0oC for 9 hours. After the cold treatment flies were transferred to fresh food vials and kept at 2oC for a 2-hour recovery period. Flies were then flash frozen in liquid nitrogen and stored at -80oC prior to RNA preparations.

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.