Abstract
Alternative polyadenylation (APA) diversifies the 3' termini of a majority of mRNAs in most eukaryotes, and is consequently inferred to have substantial consequences for the utilization of post-transcriptional regulatory mechanisms. Since conventional RNA-sequencing methods do not accurately define mRNA termini, a number of protocols have been developed that permit sequencing of the 3' ends of polyadenylated transcripts (3'-seq). We present here our experimental protocol to generate 3'-seq libraries using a dT-priming approach, including extensive details on considerations that will enable successful library cloning. We pair this with a set of computational tools that allow the user to process the raw sequence data into a filtered set of clusters that represent high-confidence functional polyadenylation sites. The data are single-nucleotide resolution and quantitative, and can be used for downstream analyses of APA.