Embryos were aged 2.5 hours, fixed in methanol, and three individual embryos at mitotic cycle 14 (mid-cellularization) were chosen under a light microscope. Each single embryo was embedded in a cryoprotecting gel, flash frozen and cut into six 60 um transverse sections along the anterior-posterior axis. The horizontal position of the blade was adjusted between slices to prevent carry-over from previous slices. Total RNA (about 15 ng) from each slice was isolated by Trizol with 20 ng glycogen carrier. This small amount of RNA was then mixed with about 100 ng of carrier total RNA from a single embryo of a distantly related Drosophila species (D. persimilis, D. willistoni or D. mojavensis).

Libraries were made according to a modified TruSeq mRNA protocol from Illumina. First, all reactions were prepared at half-volume sizes to increase relative sample concentration. Second, after AmpureXP cleanup steps, all of the resuspended sample was recovered (instead of leaving 1-3 ul as recommended). Third, only 13 cycles of PCR amplification were performed (instead of 15 cycles) to minimize PCR bias.

Illumina HiSeq 2000, 50 nt paired-end reads.

Reads were mapped using TopHat v2.0.6 to a combination of FlyBase reference genomes (version FB2012_05) for D. melanogaster and the appropriate carrier species genomes. Reads were assigned to either the D. melanogaster or carrier genomes if there were at least 4 positions per read to prefer one species over the other. Only reads that mapped to D. melanogaster were used to generate transcript abundances in Cufflinks.