Pelechano V, Wilkening S, Järvelin AI, Tekkedil MM, Steinmetz LM
Meth. Enzymol. 513 (-) 271-296 [2012-08-30; online 2012-08-30]
Alternative polyadenylation site usage gives rise to variation in 3' ends of transcripts in diverse organisms ranging from yeast to human. Accurate mapping of polyadenylation sites of transcripts is of major biological importance, since the length of the 3'UTR can have a strong influence on transcript stability, localization, and translation. However, reads generated using total mRNA sequencing mostly lack the very 3' end of transcripts. Here, we present a method that allows simultaneous analysis of alternative 3' ends and transcriptome dynamics at high throughput. By using transcripts produced in vitro, the high precision of end mapping during the protocol can be controlled. This method is illustrated here for budding yeast. However, this method can be applied to any natural or artificially polyadenylated RNA.