Poly(A)-tail-primed sequencing (2P-Seq) is designed to quantify mRNA stability and translational efficiency by characterizing 3’UTR isoforms (Spies et al., 2013). First, poly(A)+ mRNAs are isolated from total RNA and partially digested by RNase T1 to cleave the non-UTR regions. Poly (A)+ mRNA fragments are reverse-transcribed with a 20T-VN primer, which contains a stretch of 20 Ts and a VN anchor (N represents a fully degenerate base, and V is any base except for T). The resultant cDNAs are circularized and PCR-amplified using barcoded primers. The cDNA is sequenced with a custom primer ending with 20 Ts. To avoid 2P tags enriched for adenosine, analyses should be restricted to 2P tags mapping to within 20 bases of 3P-Seq_annotated poly(A) sites.
- Quantifies mRNA stability and translational efficiency
- Accurately measures half-lives and length of each UTR isoform
- 3P-Seq datasets are needed to limit results specifically to poly(A) sites
- Poly(A) primer can misprime with A-rich regions elsewhere in the mRNA
Illumina Library prep and Array Kit Selector
Kim M., You B. H. and Nam J. W. Global estimation of the 3′ untranslated region landscape using RNA sequencing. Methods. 2015;83:111-117
Spies N., Burge C. B. and Bartel D. P. 3′ UTR-isoform choice has limited influence on the stability and translational efficiency of most mRNAs in mouse fibroblasts. Genome Res. 2013;23:2078-2090