2P-Seq
Poly(A)-Tail-Primed Sequencing
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.
Advantages:
- Quantifies mRNA stability and translational efficiency
- Accurately measures half-lives and length of each UTR isoform
Disadvantages:
- 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
Reagents:
Illumina Library prep and Array Kit Selector
Reviews:
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
References:
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