Total RNA was primed for first strand cDNA synthesis using a random N15 primer in the presence of trehalose and sorbitol. The RNA/cDNA hybrid was purified, then capped RNA was biotinylated and purified using streptavidin beads. The first strand cDNA was ligated to another primer at the 5' end for second strand cDNA synthesis. The resulting double-stranded cDNA was cleaved using EcoP15I and a second linker was ligated to the 3' end of the ds-cDNA products. 5'-CAGE tags were purified by streptavidin then amplified by PCR.
Library was sequenced using Illumina platform.
Read length (bases):36 (27nt after 9nt adapter trimmed)
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/).
CAGE tags identify a diverse population of RNA elements. These include the 5' ends of capped transcripts; 5' ends of some uncapped transcripts including mitochondrial transcripts and rRNAs, which are very abundant in total RNA; and 5' ends of transcript fragments that tend to be associated with 3' UTRs.
CAGE peaks within 3' UTRs appear to be associated with cytoplasmic transcript degradation products, and not independent promoters.
