A novel method for genome-wide profiling of poly(A)-tail lengths

Polyadenylation at the 3' end of eukaryotic messenger RNAs enhances mRNA stability and translational efficiency. Global analysis for poly(A) tail lengths may shed lights on various aspects of gene regulation studies. Two NGS-based methods have been introduced for genome-wide poly(A) profiling, and they have shown human poly(A) profiles with shorter than previously conceived tail lengths. However, both methods are technically challenging and difficult to be repeated or widely adapted. Here we present a more straightforward method for poly(A) profiling. Poly(A)-seq performed on Illumina NextSeq 500 produces single-end 300 nt reads that covers the entirety of poly(A) tails, and poly(A) lengths can be directly calculated from base call data. With Poly(A)-seq we report that the global poly(A) lengths of several human cell lines may be longer than previously reported. We also show that the size selection step during Poly(A)-seq library preparation may greatly affect the sequencing profile, and thus cautions should be taken for comparisons between samples. As a convenient tool, we hope wide applications of Poly(A)-seq helps to bring better understanding of poly(A) tail properties and functions. Overall design: The poly(A) length profiles of human HeLa (wild-type and siPTB), HEK293T and HCT116 cells were examined.