Flies were raised in incubators with no photoperiod at 22 C and 60% humidity. Adults were aged 5-7 days post-eclosion and sorted by sex.

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 (300bp +/- 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).