Canton-S feeding third instar larvae were reared at 22oC on a 12:12hr light:dark regime on standard Drosophila diet. Tissues were isolated by hand dissection from equal numbers of male and female flies (except in the case of gonads). Multiple samples were pooled to obtain enough total RNA.

Total RNA was extracted by RNEasy Micro kit (Invitrogen). 2ug of total RNA was used for labeled cRNA synthesis, according to Affymetrix protocol.

Following fragmentation, 10 ug of cRNA were hybridized for 16 hr at 45[o]C on GeneChip Drosophila Genome 2 Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 450. GeneChips were scanned using the GeneChip scanner 3000 7G.

The data were analyzed with GCOS1.4 using Affymetrix default analysis settings and global scaling as normalization method. The trimmed mean target intensity of each array was arbitrarily set to 100. For each tissue, the entire procedure was repeated 4 times, to create 4 independent replicates.

Quantitation of expression at gene level: FlyAtlas provided mean coverage levels for Affy2 probe sets, and FlyBase intersected these probe sets with the exons of all FlyBase genes to associate each probe set with one or more FlyBase genes. These values were distributed into five expression level bins for further analysis.