TAIL-Seq
Poly(A) Tail Sequencing
TAIL-Seq focuses on sequencing the very ends of mRNA molecules (3′-UTRs and poly(A) tail regions) to explore their role in mRNA half-life, stability, their impact on translational efficiency, and to discover other aspects surrounding 3′-terminome function (Chang et al., 2014). Ribosomal RNA (rRNA) is first removed from total RNA by affinity-based depletion. After purification, mRNAs are ligated to biotinylated 3′ adapters prior to RNase T1 fragmentation and purified further by streptavidin pull-down. The 5′ ends are phosphorylated and size-selected for 500_1000 nt fragments to prevent short noncoding RNA (ncRNA) fragments from contaminating the sequence data. The phosphorylated 5′ ends are ligated to 5′ adapters, reverse-transcribed, PCR-amplified, and sequenced. TAIL-Seq uses a unique paired-end run system to correlate the genes corresponding to the 3′-end sequence reads. By separating the paired-end reads, read 1 sequences 52 nt from the 5′ end of the fragment to map the genome and identify transcripts, while read 2 sequences 251 nt from the 3′ end specifically for sequence determination.
Advantages:
- Quantifies poly(A) tail length on mRNA samples with high accuracy using a special fluorescence analysis method
- Eliminates bias toward long poly(A) because it does not use oligo(dT) enrichment
- Able to detect modified 3′ ends, unlike poly(A)-tail length profiling by sequencing (PAL-Seq) (Nussbacher et al., 2015)
Disadvantages:
- PCR amplification is unfavorable for homopolymeric sequences
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
Hrdlickova R., Toloue M. and Tian B. RNA-Seq methods for transcriptome analysis. Wiley Interdiscip Rev RNA. 2017;8:n/a-n/a
Bangru S. and Kalsotra A. Advances in analyzing RNA diversity in eukaryotic transcriptomes: peering through the Omics lens. F1000Research. 2016;5:2668
Viegas S. C., Silva I. J., Apura P., Matos R. G. and Arraiano C. M. Surprises in the 3′-end: ‘U’ can decide too! FEBS J. 2015;282:3489-3499
References:
Zuber H., Scheer H., Ferrier E., et al. Uridylation and PABP Cooperate to Repair mRNA Deadenylated Ends in Arabidopsis. Cell Rep. 2016;14:2707-2717
Park J. E., Yi H., Kim Y., Chang H. and Kim V. N. Regulation of Poly(A) Tail and Translation during the Somatic Cell Cycle. Mol Cell. 2016;62:462-471
Lapointe C. P., Wilinski D., Saunders H. A. and Wickens M. Protein-RNA networks revealed through covalent RNA marks. Nat Methods. 2015;12:1163-1170
Related
History: TAIL-Seq
Revision by sbrumpton on 2017-06-21 07:50:24 - Show/Hide
Poly(A) Tail Sequencing
TAIL-Seq focuses on sequencing the very ends of mRNA molecules (3'-UTRs and poly(A) tail regions) to explore their role in mRNA half-life, stability, their impact on translational efficiency, and to discover other aspects surrounding 3'-terminome function (Chang et al., 2014). Ribosomal RNA (rRNA) is first removed from total RNA by affinity-based depletion. After purification, mRNAs are ligated to biotinylated 3' adapters prior to RNase T1 fragmentation and purified further by streptavidin pull-down. The 5' ends are phosphorylated and size-selected for 500_1000 nt fragments to prevent short noncoding RNA (ncRNA) fragments from contaminating the sequence data. The phosphorylated 5' ends are ligated to 5' adapters, reverse-transcribed, PCR-amplified, and sequenced. TAIL-Seq uses a unique paired-end run system to correlate the genes corresponding to the 3'-end sequence reads. By separating the paired-end reads, read 1 sequences 52 nt from the 5' end of the fragment to map the genome and identify transcripts, while read 2 sequences 251 nt from the 3' end specifically for sequence determination.
Advantages:- Quantifies poly(A) tail length on mRNA samples with high accuracy using a special fluorescence analysis method
- Eliminates bias toward long poly(A) because it does not use oligo(dT) enrichment
- Able to detect modified 3' ends, unlike poly(A)-tail length profiling by sequencing (PAL-Seq) (Nussbacher et al., 2015)
Disadvantages:- PCR amplification is unfavorable for homopolymeric sequences
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;38:226-236Hrdlickova R., Toloue M. and Tian B. RNA-Seq methods for transcriptome analysis. Wiley Interdiscip Rev RNA. 2017;8:n/a-n/aBangru S. and Kalsotra A. Advances in analyzing RNA diversity in eukaryotic transcriptomes: peering through the Omics lens. F1000Research. 2016;5:2668Viegas S. C., Silva I. J., Apura P., Matos R. G. and Arraiano C. M. Surprises in the 3'-end: 'U' can decide too! FEBS J. 2015;282:3489-3499References:Zuber H., Scheer H., Ferrier E., et al. Uridylation and PABP Cooperate to Repair mRNA Deadenylated Ends in Arabidopsis. Cell Rep. 2016;14:2707-2717Park J. E., Yi H., Kim Y., Chang H. and Kim V. N. Regulation of Poly(A) Tail and Translation during the Somatic Cell Cycle. Mol Cell. 2016;62:462-471Lapointe C. P., Wilinski D., Saunders H. A. and Wickens M. Protein-RNA networks revealed through covalent RNA marks. Nat Methods. 2015;12:1163-1170