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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.
Researchers from the University of Texas, Medical Branch recently reported a click-chemistry based method for generating RNAseq libraries called ‘ClickSeq’. Here, they adapt this method to direct the cDNA synthesis specifically toward the 3΄UTR/poly(A) tail junction of cellular RNA. With this novel approach, the researchers 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, they 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, they utilize PAC-seq to quantify and analyze APA events regulated by CFIm25 illustrating how this technology can be harnessed to identify alternatively polyadenylated RNA.
Schematic overview of Poly(A)ClickSeq (PAC-seq)
(A) RT-PCR is launched from a non-anchored Poly(T) primer containing a portion of the Illumina p7 adaptor. RT-PCR is performed in the presence of AzATP, AzGTP and AzCTP, but not AzTTP, thus only allowing chain termination to occur upstream of the poly(A) tail in the 3΄UTR. (B) 3΄-Azido-blocked cDNA fragments are ‘click-ligated’ to 5΄-hexynyl–functionalised DNA oligos containing the p5 Illumina adaptor. This yields triazole-linked ssDNA which can be PCR-amplified using primers to the p5 and p7 Illumina adaptors. (C) The cDNA library is analysed by gel electrophoresis and should consist of a smear of DNA products centered ∼200–300 bp. Appropriate cDNA fragment sizes are cut out of the gel and purified to yield a final library. (D) The final library consists of DNA fragments containing the Illumina p5 adaptor, a portion of the 3΄UTR, a stretch of As derived from both the RNA template and the poly(T) primer, and finally the p7 Illumina Indexing primer.
