Abstract
Poly(A) tail metabolism is critical for various biological processes, including early embryogenesis and cell differentiation. While traditional biochemical methods to measure poly(A) tail length allow for the study of selected transcripts, the advent of long-read sequencing technologies enabled the development of simple and robust protocols to measure poly(A) tail length at the transcriptome level. Here, we describe a direct RNA sequencing protocol to capture poly(A) tail terminal additions based on the splint ligation of barcoded oligos compatible with terminal guanylation and uridylation. We cover how to prepare the libraries and perform the bioinformatics analysis to simultaneously determine the length of the transcripts’ poly(A) tails and detect the presence of terminal guanylation and uridylation.
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Acknowledgments
This study was supported by the research project ZIA ES103355 funded by the Division of Intramural Research of the National Institute of Environmental Health Sciences, National Institutes of Health, which was awarded to MM. The authors would like to thank Yolanda L. Jones for her help in editing the manuscript.
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Gupta, A., Papas, B.N., Baptissart, M., Morgan, M. (2024). Quantification of Poly(A) Tail Length and Terminal Modifications Using Direct RNA Sequencing. In: Valkov, E., Goldstrohm, A.C. (eds) Deadenylation. Methods in Molecular Biology, vol 2723. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3481-3_15
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DOI: https://doi.org/10.1007/978-1-0716-3481-3_15
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Publisher Name: Humana, New York, NY
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Online ISBN: 978-1-0716-3481-3
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