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.


  • 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)


  • PCR amplification is unfavorable for homopolymeric sequences


Illumina Library prep and Array Kit Selector


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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


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

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