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Paired-End Analysis of Transcript start sites (PEAT)
We developed the Paired-End Analysis of TSSs (PEAT) strategy to characterize the landscape of transcription initiation in eukaryotes. This protocol is designed to harnesses the paired-end capability of the Illumin/Solexa platform. Similar to CAGE, our protocol is designed to sequence the 5' end of all capped transcripts in a eukaryotic cell. Along with that information PEAT provides a short sequence approximately 270nt downstream of the 5' sequence within the same transcript. Compared to the CAGE method, our strategy has several advantages in experimental design. A circularization step is used to improve the overall specificity of the library contstruction by removing undesired cDNA fragments without the matched adapters; and the subsequenct rolling circle amplification (RCA) can help reduce amplification biases and erroneous bases introduced during library construction. More importantly, paired-end reads generated by the PEAT method provides us with higher alignmnet yield and accuracy as well as additional information on gene structure (e.g., linking 5' TSS tags to known genes or transcripts). We employed this strategy to monitor global TSS usage during Drosophila melanogaster embryogenesis.