Umbrella record for 12 collections that differ by developmental stage, from embryos to aged adults. Transcriptome represented as frequency of reads along genome (see GBrowse presentation; data in wiggle format). RNA junctions identified and characterized (presented in GBrowse and in RNA junction reports).
Whole body samples were homogenized in 1ml of Trizol per 50-100 mg of tissue to isolate total RNA. Polyadenylated mRNA was then isolated from 20ug total RNA using an mRNA Purification Kit (Invitrogen). Double-stranded cDNA was made using Superscript Double-Stranded cDNA Synthesis Kit (Invitrogen) and random hexamer primers. Size fractionated 200-400bp cDNAs were used for Solexa library construction (paired end layout).
The cDNA library was characterized by high-throughput sequencing (Illumina GAII).
Reads were aligned to the Drosophila reference genome (Dmel_Release_5) using BLAT. The BLAT ooc file was prepared with -repMatch=128 and otherwise default parameters were used. Paired reads were parsed to disambiguate multiple alignment locations if one of the two pairs mapped uniquely or the pair agreed on a unique mapping location. Reads with unaligned mates or pairs which did not agree were treated as single-end reads. Only reads with unique alignment location were considered for analyses.
A small number of junctions predicting very small and very long introns may be mispredictions and should be interpreted with care; these are currently being re-examined.
For ~1900 predicted junctions, the supporting evidence came from a methodology (Wang L, Xi Y, Yu J, Dong L, Yen L, et al. 2010 A Statistical Method for the Detection of Alternative Splicing Using RNA-Seq. PLoS ONE 5(1): e8529. doi:10.1371/journal.pone.0008529) that did not report number of supporting junction-spanning reads. Each of these ~1900 predicted junctions was supported by at least two reads, and so we have noted these by having a total junction-spanning read count of ">1". However, we do not know which developmental stages these supporting reads were in, and so all developmental stages are reported as "0".
