Adults were aged 8 days post-eclosion and sorted by sex. Collection of adult heads: frozen whole flies were placed on 10.8 x 10.8 cm ceramic plates frozen on dry ice; forceps were used to dissect 400-500 heads for subsequent RNA extraction.

mRNA was purified from total RNA using a QIAGEN Oligotex mRNA kit (Valencia, California). mRNA quality and quantity was determined by Bioanalyzer (Agilent, Santa Clara, California) and 260/280 ratio from Nanodrop ND-1000 UV spectrophotometer. Double-stranded cDNAs were made from mRNA using the Superscript II (Invitrogen) and random hexamer primers (Invitrogen). DNA fragment libraries were prepared for sequencing by addition adaptor oligonucleotides; cDNA templates were size-selected by gel purification (200bp +/- 25bp), then amplified by PCR using oligo-specific primers and Phusion polymerase. The amplified product was purified using a QIAquick column.

Read length (bases):75

Only Illumina GAII data was used. Raw reads were trimmed to 75 nt before mapping (shorter reads were removed) and mapped using TopHat (v2.0.8b) with parameters: -g 1 -r 150 --solexa1.3-quals (except for data from head samples where parameters were -g 1 -r 150).

Strain used: line MV2-25 Baylor sequencing strain, obtained from the Drosophila Species Stock Center (DSSC).