For the RE cDNA library, embryos at 0-22 hr AEL were collected from an isogenic y; cn bw sp (iso-1) strain.
RNA was polyA+ selected twice (RNA made by L. Hong). cDNA was synthesized by priming with the oligo(dT) primer adapter (5'-GAGAGAGAGAGGATCCAATACTGGAGAGTTTTTTTTTTTTTTTTVN-3'). The first strand was synthesized in presence of trehalose, which increases the full-length cDNA synthesis (Carninci, P. et al. 1998. Proc. Natl. Acad. Sci. U.S.A. 95(2): 520-524; Carninci, P. et al. 1999. Methods Enzymol. 303: 19-44). Full-length cDNA was selected with the biotinylated cap-trapper (Carninci, P. et al. 1996. Genomics 37(3): 327-336). A linker was then ligated to the single-strand cDNA following the published protocol (Shibata, Y., et al. 2001. Biotechniques 30(6): 1250-1254). Subsequently, the cDNA was normalized by using RoT=1.0 as published (Carninci, P., et al. 2000. Genome Res. 10(10): 1617-1630). Second strand cDNA was primed with the (5'-AGAGAGAGAGCTCGAGCTCTAATAAGGTGACACTATAGAACCA-3') primer. After restriction digestion of the hemimethylated cDNA with BamHI and XhoI, the cDNA was cloned in the lambda FLC-I vector. Subsequently, the library was bulk-excised into pFLC-I plasmid as described (Carninci, P., et al. 2001. Genomics 77(1-2): 79-90). cDNAs were transformed into DH5-alpha TonA strain.
The 9-nt barcode linker sequence was removed, and the 27-nt CAGE reads representing capped 5’ transcript ends were aligned to the D. melanogaster genome using StatMap (http://www.statmap-bio.org/).
modENCODE Transcription Start Sites
Core promoter sequence motifs are differentially enriched in the peaked and broad classes of promoters.
Genes with peaked promoters have a marked and highly significant tendency to be expressed in spatially and temporally restricted patterns, and genes with broad promoters do not.
CAGE peaks within 3' UTRs appear to be associated with cytoplasmic transcript degradation products, and not independent promoters.