Application and design considerations for 3′-end sequencing using click-chemistry

Madeline K. Jensen, Nathan D. Elrod, Hari Krishna Yalamanchili, Ping Ji, Ai Lin, Zhandong Liu, Eric J. Wagner

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

Over the past 15 years, investigations into alternative polyadenylation (APA) and its function in cellular physiology and pathology have greatly expanded due to the emergent appreciation of its key role in driving transcriptomic diversity. This growth has necessitated the development of new technologies capable of monitoring cleavage and polyadenylation events genome-wide. Advancements in approaches include both the creation of computational tools to re-analyze RNA-seq to identify APA events as well as targeted sequencing approaches customized to focus on the 3′-end of mRNA. Here we describe a streamlined protocol for polyA-Click-seq (PAC-seq), which utilizes click-chemistry to create mRNA 3′-ends sequencing libraries. Importantly, we offer additional considerations not present in our previous protocols including the use of spike-ins, unique molecular identifier primers, and guidance for appropriate depth of PAC-seq. In conjunction with the companion chapter on PolyA-miner (Yalamanchili et al., 2021) to computationally analyze PAC-seq data, we provide a complete experimental pipeline to analyze mRNA 3′-end usage in eukaryotic cells.

Original languageEnglish (US)
Title of host publicationMethods in Enzymology
PublisherAcademic Press Inc.
DOIs
StateAccepted/In press - 2021

Publication series

NameMethods in Enzymology
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

Keywords

  • 3′-end sequencing
  • Alternative polyadenylation
  • PAC-seq
  • PolyA-miner

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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