PAT-Seq: A Method for Simultaneous Quantitation of Gene Expression, Poly(A)-Site Selection and Poly(A)-Length Distribution in Yeast Transcriptomes

Angavai Swaminathan, Paul F. Harrison, Thomas Preiss, Traude H. Beilharz

Research output: Chapter in Book/Report/Conference proceedingChapter (Book)Otherpeer-review

1 Citation (Scopus)

Abstract

Next-generation sequencing (NGS) and its application to RNA (RNA-seq) has opened up multiple aspects of RNA processing to deep transcriptome-wide analysis at nucleotide resolution. This has been useful in delineating the transcribed areas of the genome, and in quantitation of RNA isoforms. Such isoforms can diversify the regulatory repertoire of mRNAs. For example, the 3′-end of mRNA can vary in two important ways, in the position chosen for cleavage and polyadenylation, and in the length of the poly(A)-tail. Accordingly, the step-up in resolution made possible by NGS has revealed an unexpectedly high level of alternative polyadenylation (APA). Moreover, it has massively simplified the transcriptome-wide detection of poly(A)-tail length changes. Here we present our approach to the study of 3′-end dynamics using a 3′-focused RNA-seq method called PAT-seq (for poly(A)-test sequencing). The approach records gene expression, APA, and poly(A)-tail changes between transcriptomes to reveal complex interplay between transcriptional and posttranscriptional control mechanisms.

Original languageEnglish
Title of host publicationYeast Systems Biology
Subtitle of host publicationMethods and Protocols
EditorsStephen G. Oliver, Juan I. Castrillo
Place of PublicationNew York, NY
PublisherHumana Press
Chapter9
Pages141-164
Number of pages24
Edition2nd
ISBN (Electronic)9781493997367
ISBN (Print)9781493997350
DOIs
Publication statusPublished - 2019

Publication series

NameMethods in Molecular Biology
Volume2049
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

Keywords

  • Alternative polyadenylation
  • Digital gene expression
  • ePAT
  • PAT assay
  • Poly(A)-tail
  • RNA-seq

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