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Polyadenylation Site-Based Analysis of Transcript Expression by 3′READS+

  • Dinghai Zheng
  • Bin Tian
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1648)

Abstract

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.

Key words

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

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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Department of Microbiology, Biochemistry and Molecular GeneticsRutgers New Jersey Medical School, Rutgers Cancer Institute of New JerseyNewarkUSA

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