Embryos were collected in one-hour windows on grape-agar plates after two two-hour prelays and aged appropriately. Embryos were dechorionated and snap frozen in liquid nitrogen.
Total RNA was isolated using the RNAdvance Tissue kit (Agencourt) and treated with DNase I. RNA quality was checked on a Bioanalyzer (Agilent). 5'-monophosphate RNA species, mainly ribosomal RNA, were depleted by TEX digest (Epicentre). Reverse transcriptase was done under conditions that promote "template switching" to preferentially add linkers to the 5' ends of 5'-complete cDNAs for second strand synthesis. 5'-capped RNA molecules were then biotinylated and pulled down to serve as templates for second strand cDNA synthesis. cDNA was PCR amplified and DNA quality was confirmed on a DNA HS Bioanalyzer chip.
Three multiplexed libraries were prepared: one for embryos (24 samples), one for larvae and pupae (10 samples) and one for adults (two samples).
Read length (bases): 101
Samples were processed as multiplexed libraries: one that pooled all 24 embryonic samples, one that pooled all 10 larval and pupal samples, and one that pooled both adult fly samples.
Total reads: 3,732,348
Read length (bases): 76
Uniquely mapped reads: 2,922,415
Total reads: 296,251,429
Uniquely mapped reads: 228,277,829
Sequences corresponding to library identification barcodes and primers were trimmed. Reads were mapped with STAR. All uniquely mapping reads were kept. For multiply mapping reads, if all alignments started within an annotated transposon and overlapped the same gene annotation, the alignment starting in the closest transposon was selected. Transcript annotations were obtained from FlyBase (release 5.32).