title: RNA-seq of Drosophila melanogaster: adult male head
design: mRNA from flash-frozen fly heads was purified using Dynbeads mRNA DIRECT Micro kit (Invitrogen, USA) with slight modifications.  Heads were homogenized in 200 uL Lysis Binding Buffer using a GenoGrinder for 2 minutes.  0.5 uL of a 1/20 dilution of ERCC spike (Invitrogen, USA) was added to the lysate prior to combining with the magnetic beads. Beads were washed and mRNA was eluted and rebound to the beads, rewashed. Beads were collected and suspended in first strand buffer (Invitrogen, USA). Supernatant containing fragmented mRNA was transferred to a new tube first strand cDNA synthesis using random hexamer primers (Invitrogen, USA) was performed. After reverse transcription, second strand synthesis was carried out immediately and cDNA was subsequently purified with AMPure XP (Beckman Coulter, USA). Illumina RNAseq libraries were prepared by Rapid Genomics (http://rapid-genomics.com/home/) using dual barcodes. Sequencing was performed using the Illumina HiSeq 2500 as either 2150bp or 250bp reads.  There were between 8.2 and 16.3 million reads per sample in total (~11.5 million reads on average, 1,065 million reads in total).