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

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