Poly(A)-ClickSeq: click-chemistry for next-generation 3΄-end sequencing without RNA enrichment or fragmentation

Nucleic Acids Res. 2017 Jul 7;45(12):e112. doi: 10.1093/nar/gkx286.


The recent emergence of alternative polyadenylation (APA) as an engine driving transcriptomic diversity has stimulated the development of sequencing methodologies designed to assess genome-wide polyadenylation events. The goal of these approaches is to enrich, partition, capture and ultimately sequence poly(A) site junctions. However, these methods often require poly(A) enrichment, 3΄ linker ligation steps, and RNA fragmentation, which can necessitate higher levels of starting RNA, increase experimental error and potentially introduce bias. We recently reported a click-chemistry based method for generating RNAseq libraries called 'ClickSeq'. Here, we adapt this method to direct the cDNA synthesis specifically toward the 3΄UTR/poly(A) tail junction of cellular RNA. With this novel approach, we demonstrate sensitive and specific enrichment for poly(A) site junctions without the need for complex sample preparation, fragmentation or purification. Poly(A)-ClickSeq (PAC-seq) is therefore a simple procedure that generates high-quality RNA-seq poly(A) libraries. As a proof-of-principle, we utilized PAC-seq to explore the poly(A) landscape of both human and Drosophila cells in culture and observed outstanding overlap with existing poly(A) databases and also identified previously unannotated poly(A) sites. Moreover, we utilize PAC-seq to quantify and analyze APA events regulated by CFIm25 illustrating how this technology can be harnessed to identify alternatively polyadenylated RNA.

MeSH terms

  • 3' Untranslated Regions*
  • Animals
  • Base Sequence
  • Click Chemistry / methods*
  • DNA, Complementary / genetics
  • DNA, Complementary / metabolism
  • Databases, Genetic
  • Drosophila melanogaster / genetics
  • Drosophila melanogaster / metabolism
  • Gene Expression Profiling
  • Gene Library
  • HeLa Cells
  • High-Throughput Nucleotide Sequencing*
  • Humans
  • Molecular Sequence Annotation
  • Poly A / analysis*
  • Poly A / genetics
  • Poly A / metabolism
  • Polyadenylation
  • RNA, Messenger / analysis*
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • Sequence Analysis, RNA
  • Transcriptome*
  • mRNA Cleavage and Polyadenylation Factors / genetics
  • mRNA Cleavage and Polyadenylation Factors / metabolism


  • 3' Untranslated Regions
  • DNA, Complementary
  • RNA, Messenger
  • mRNA Cleavage and Polyadenylation Factors
  • Poly A