RNA recovered from 5 pupal collections representing successive stages (daily collections, 1-5 days after third larval instar) was mixed in equimolar amounts.

Whole body samples were homogenized in 1ml of Trizol per 50-100 mg of tissue to isolate total RNA. Polyadenylated mRNA was then isolated from 20ug total RNA using an mRNA Purification Kit (Invitrogen). Double-stranded cDNA was made using Superscript Double-Stranded cDNA Synthesis Kit (Invitrogen) and random hexamer primers. Size fractionated 200-400bp cDNAs were used for Solexa library construction (paired end layout).

Reads were aligned to the Drosophila reference genome (Dmel_Release_5) using BLAT. The BLAT ooc file was prepared with -repMatch=128 and otherwise default parameters were used. Paired reads were parsed to disambiguate multiple alignment locations if one of the two pairs mapped uniquely or the pair agreed on a unique mapping location. Reads with unaligned mates or pairs which did not agree were treated as single-end reads. Only reads with unique alignment location were considered for analyses.