Embryos were fixed in methanol and individual embryos were selected under a light microscope at seven different time points based on morphology: stage 2, stage 4 and five time points within stage 5. Each single embryo was embedded in a cryoprotecting gel, flash frozen and cut into 25 um transverse sections along the anterior-posterior axis, yielding between 10 and 15 contiguous, usable slices. 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 carrier total RNA from yeast S. cerevisiae or T. delbruckii.

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.