3P-Seq
3P-Seq
Poly(A)-position profiling by sequencing (3P-Seq) is used to identify 3'-UTRs in mRNA. Poly(A) selection is used to isolate mRNA from total RNA, and biotinylated-splint primers are annealed and splint-ligated to the end of the mRNA poly(A) tail. The RNA-primer complex is partially digested by RNase T1, bound to streptavidin, and washed to purify the 3' fragments. Primers corresponding to the 3'end of the poly(A) tail are annealed and reverse-transcribed with deoxythymidine triphosphate (dTTP) as the only dNTP to generate a cDNA strand complementary to the poly(A) tail. The biotin-bound poly(A) RNA fragments are released by RNase H digestion and purified. Next, P7 and P5 adapters are attached to the RNA fragments, and they are reverse-transcribed to generate cDNA fragments.
Pros:
- Reliable for UTR isoform discoveries
- Prevents polymerase slippage by using splint-ligation primer in poly(A) capture
- Prevents internal priming and is specific to 3' ends of poly(A) RNAs
Cons:
- Needs large amounts of RNA
- Technically challenging to perform
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- 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