Fly stocks were reared in bottles at 24oC and males were collected immediately after eclosion, then frozen on dry ice.
Frozen samples were homogenized and extracted using the TRIzol reagent protocol (Invitrogen). RNA was purified on an RNeasy spin column (Qiagen), and DNase treated. Polyadenylated RNAs were purified from total RNA extracts via oligo(dT) binding, using standard Illumina protocol. The poly(A)+ RNA was fragmented using divalent cations under elevated temperature, following by first and second strand cDNA synthesis primed with random hexamers. The cDNA fragments were end-repaired using T4 DNA polymerase and Klenow DNA polymerase, and phosphorylated at their 5' ends with T4 polynucleotide kinase. After adding A bases to the 3' end of the DNA fragments, Illumina adaptor oligonucleotides were ligated to the ends and ~ 300 bp fragments were isolated from an agarose gel, enriched by PCR amplification, and gel-purified again.
Read length (bases):76
The samples were quantitated using a Nanodrop, and loaded onto a flow cell for cluster generation and sequenced on an Illumina Genome Analyzer II using either single read or paired end protocols (Illumina).
RNA-Seq reads were aligned to databases of predicted splice junctions and to gene models using TopHat, STAR and BLAT. A number of criteria were used to distinguish true splice junctions from false positives. First, only reads with a minimum of 6nt overhang across a junction were considered. Second, an entropy score (which reflects the number of offset reads) was calculated for each splice junction, and only junctions with entropy score >2 in at least two biological samples were considered. The set of junctions was filtered further to exclude junctions when intron length less than 41 nt, junctions that join parologous genes and non-canonical splice junctions not supported by other experimental support.