Accurate mapping of cleavage and polyadenylation sites by 3' region extraction and deep sequencing.

Deep sequencing of RNA (RNA-seq) is becoming a standard method to study gene expression. While RNA-seq reads cover most regions of an mRNA sequence, they are often depleted in the 3' end region, making them less amenable for mapping the cleavage and polyadenylation site (pA). A major problem in identification of pA is mispriming at internal A-rich regions and oligo(A) tails when an oligo(dT) primer is used for reverse transcription or sequencing. We recently developed a method named 3' region extraction and deep sequencing (3'READS), which completely addresses mispriming issues and is straightforward to use. The method accurately maps pAs and allows quantitative analysis of alternative cleavage and polyadenylation (APA) isoforms and gene expression.