Fly stocks were reared in bottles at 24oC. Flies were collected immediately after eclosion and aged for 29 days. Males were selected 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).
To identify potential sites of RNA editing, short poly(A)+ RNA-Seq reads that mapped to annotated transcripts were compared to the reference genome to find instances in which a 'G' aligned to an 'A' (for plus strand transcripts) or a 'C' aligned to a 'T' (for minus strand). The first or last six bases of reads were not examined because they were easily mismapped to introns rather than across splice junctions. Only positions with at least five reads showing a substitution from any single developmental stage were considered further. A dataset of potential editing sites with the number of edited reads and the number of non-edited reads for each sample was created. To prevent a polymorphism in the sequenced strain from being reported as editing, at least 100 reads having an exact match to the genome were necessary for further consideration of a potential editing site. Only those sites with at least 5% of reads in at least two independent adult samples showing evidence of editing are reported.