3P-Seq
Poly(A)-Position Profiling by Sequencing
Poly(A)-position profiling by sequencing (3P-Seq) is used to identify 3′-UTRs in mRNA (Jan et al., 2011). 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.
Advantages:
- 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
Disadvantages:
- Requires large amounts of RNA
- Technically challenging to perform
Reagents:
Illumina Library prep and Array Kit Selector
Reviews:
Nussbacher J. K., Batra R., Lagier-Tourenne C. and Yeo G. W. RNA-binding proteins in neurodegeneration: Seq and you shall receive. Trends Neurosci. 2015;38:226-236
References:
Lakshmanan V., Bansal D., Kulkarni J., et al. Genome-Wide Analysis of Polyadenylation Events in Schmidtea mediterranea. G3 (Bethesda). 2016;
Hezroni H., Koppstein D., Schwartz M. G., Avrutin A., Bartel D. P. and Ulitsky I. Principles of long noncoding RNA evolution derived from direct comparison of transcriptomes in 17 species. Cell Rep. 2015;11:1110-1122
Jambor H., Surendranath V., Kalinka A. T., Mejstrik P., Saalfeld S. and Tomancak P. Systematic imaging reveals features and changing localization of mRNAs in Drosophila development. Elife. 2015;4:
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
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History: 3P-Seq
Revision by sbrumpton on 2017-06-21 07:50:23 - Show/Hide
Poly(A)-Position Profiling by Sequencing
Poly(A)-position profiling by sequencing (3P-Seq) is used to identify 3'-UTRs in mRNA (Jan et al., 2011). 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.
Advantages:- 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
Disadvantages:- Requires large amounts of RNA
- Technically challenging to perform
Reagents:Illumina Library prep and Array Kit SelectorReviews:Nussbacher J. K., Batra R., Lagier-Tourenne C. and Yeo G. W. RNA-binding proteins in neurodegeneration: Seq and you shall receive. Trends Neurosci. 2015;38:226-236References:Lakshmanan V., Bansal D., Kulkarni J., et al. Genome-Wide Analysis of Polyadenylation Events in Schmidtea mediterranea. G3 (Bethesda). 2016;Hezroni H., Koppstein D., Schwartz M. G., Avrutin A., Bartel D. P. and Ulitsky I. Principles of long noncoding RNA evolution derived from direct comparison of transcriptomes in 17 species. Cell Rep. 2015;11:1110-1122Jambor H., Surendranath V., Kalinka A. T., Mejstrik P., Saalfeld S. and Tomancak P. Systematic imaging reveals features and changing localization of mRNAs in Drosophila development. Elife. 2015;4: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