Location: Children's Nutrition Research CenterTitle: Application and design considerations for 3'-end sequencing using click-chemistry
|JENSEN, MADELINE - University Of Texas Medical Branch|
|ELROD, NATHAN - University Of Texas Medical Branch|
|YALAMANCHILI, HARI - Children'S Nutrition Research Center (CNRC)|
|JI, PING - University Of Texas Medical Branch|
|LIN, AI - University Of Texas Medical Branch|
|LIU, ZHANDONG - Baylor College Of Medicine|
|WAGNER, ERIC - University Of Texas Medical Branch|
Submitted to: Methods in Enzymology
Publication Type: Book / Chapter
Publication Acceptance Date: 5/16/2021
Publication Date: 7/2/2021
Citation: Jensen, M.K., Elrod, N.D., Yalamanchili, H.K., Ji, P., Lin, A., Liu, Z., Wagner, E.J. 2021. Application and design considerations for 3'-end sequencing using click-chemistry. In: Tian, B. editor. Methods in Enzymology. 1st edition. Cambridge, MA: Academic Press. p. 1-23. https://doi.org/10.1016/bs.mie.2021.03.012.
Interpretive Summary: Researchers are expanding the horizons of human health and disease research by tapping into alternative polyadenylation (APA), an under-charted mechanism that regulates gene expression. APA is about modifying the 3-prime end (3'end) of RNA strands that are transcribed from DNA. The modification consists of changing the length of a tail of adenosines, one of the RNA building blocks, at the 3'end before RNA is translated into proteins. Alternative polyadenylation regulates protein production by determining the length of the messenger RNA. The propelling appreciation of APA in driving transcriptomic diversity has resulted in the development of new technologies capable of monitoring cleavage and polyadenylation events genome-wide. Advancements in approaches include both computational tools as well as targeted sequencing approaches customized to focus on the 3'end of mRNA. We describe a streamlined protocol for polyA-Click-seq (PAC-seq), which utilizes click-chemistry to create mRNA 3'end sequencing libraries in  and a companion chapter on PolyA-miner , a computational pipeline to infer alternative poly-adenylation from 3' sequencing data. PolyA-miner comprehends all non-proximal to non-distal APA dynamics using vector projections and reflect precise gene-level 3'UTR changes. It can also effectively identify novel APA sites that are otherwise undetected when using reference-based approaches. APA is considered a major mechanism for RNA regulation that has strong relevance both in cancer and neurological diseases. This development has tremendous implications for basic research and for the potential translation of scientific findings into the clinic.
Technical 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.