Polyadenylation site-based analysis of transcript expression by 3′READS+

Dinghai Zheng, Bin Tian

Research output: Chapter in Book/Report/Conference proceedingChapter

5 Scopus citations


Deep sequencing of the 3′ end region of poly(A)+ RNA identifies the cleavage and polyadenylation site (PAS) and measures transcript abundance. However, mispriming at internal A-rich regions by the oligo-dT oligo in reverse transcription can lead to falsely identified PASs. This problem can be resolved by direct ligation of an adapter to the 3′ end of RNA. However, ligation-based methods are often inefficient. Here, we describe 3′READS+, an accurate and sensitive method for deep sequencing of the 3′ end of poly(A)+ RNA. Through partial digestion by RNase H of the poly(A) tail bound to a locked nucleic acid (LNA)/ DNA hybrid oligo, this method sequences an optimal number of terminal A’s, which balances sequencing quality and accurate identification of PAS in A-rich regions. With efficient ligation steps, 3′READS+ is amenable to small amounts of input RNA. 3′READS+ can also be readily used as a cost-effective method for gene expression analysis.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Number of pages13
StatePublished - 2017

Publication series

NameMethods in Molecular Biology
ISSN (Print)1064-3745

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Genetics


  • 3′ end sequencing
  • 3′READS+
  • Alternative cleavage and polyadenylation
  • Deep sequencing
  • RNA-seq


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