CAGE L3 12 hr Post-molt
CAGE sequencing and alignment of Dm Dev Timecourse L3_12hr 5' ends (Celniker project, Celniker subgroup)
D. melanogaster RNA was isolated from staged L3 larvae, 12 hr post-molt, and used to prepare cDNA. After biotinylating the 5' cap, full-length heteroduplexes were purified using streptavidin beads. An oligonucleotide adaptor containing the restriction site for a type III endonuclease (EcoP15I) was ligated onto the first strand cDNA, and used to prime second-strand cDNA synthesis. After cleavage with EcoP15I the 27 nt fragments were amplified and sequenced on an Illumina GA-IIx sequencer. The reads were aligned to the reference genome using StatMap.
To map transcription start sites (TSSs) genome-wide, we perform CAGE (Cap Analysis of Gene Expression). These CAGE tags are 27-nt sequences that are derived from mRNAs and other long capped transcripts in the vicinity of the 5' cap site. Their mapping onto the genome sequence defines TSSs. CAGE libraries are constructed from first-strand cDNA selected through a biotinylated cap. Second-strand synthesis is dependent upon ligation to the first-strand full-length cDNA of a primer that contains a restriction site for a type II endonuclease allowing the cleavage of 5' 27-bp tags from the resulting cDNA. These short fragments are sequenced using the Illumina GA platform.
Referenced modENCODE submissions:
Related modENCODE submissions:
Release Date: 2012-12-18