Total RNA was isolated. Gene-specific 5' RLM-RACE products were generated using the FirstChoice RLM-RACE procedure (Ambion). Primers were designed to target all FlyBase release 5.12 (October 2008) transcript models that overlap 5' ESTs from the RE (FBrf0152058) and LD (FBrf0127297) cDNA libraries. Transcripts of genes expressed in the embryo based on whole-mount RNA in situ hybridization (FBrf0205271) and literature surveys were also targeted. In all, 8570 distinct primer pairs representing 7742 genes were designed. For 1453 RACE reactions that lacked detectable product, second-round PCR conditions were used that including 5 extra amplification cycles.
Individual PCR products were pooled to create a molar-normalized mixtures of 1,440 to 2,677 products using a Tecan Genesis 2 robot. The sample pools were then concentrated to approximatley 50 ng/ul. Normalized pools of RACE products were created and either cloned and sequenced with an ABI3730 or by high-throughput sequencing using the "454 GS FLX" platform. Vector sequences were removed and the RNA adapter sequence was used to determine the orientation of the clone and was removed from the sequence. Each sequence represents a potential transcription start site and is oriented along the direction of transcription.
Clones were not preserved and are not available for distribution.