ABSTRACT Advances in RNA-sequencing methods have uncovered many aspects of RNA metabolism but are limited to surveying either the 3ʹ or 5ʹ terminus of RNAs, thus missing mechanistic aspects

that could be revealed if both ends were captured. We developed Akron sequencing (Akron-seq), a method that captures in parallel the native 5ʹ ends of uncapped, polyadenylated mRNAs and 3ʹ

ends of capped mRNAs from the same input RNA. Thus, Akron-seq uniquely enables assessment of full-length and truncated mRNAs at single-nucleotide resolution. Akron-seq involves RNA

isolation, depletion of ribosomal and abundant small capped RNAs, and selection of capped and polyadenylated mRNAs. The endogenous ends of mRNAs are marked by adaptor ligation, followed by

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PROFILING Article 25 November 2022 TRANSCRIPTOME-WIDE PROFILING AND QUANTIFICATION OF _N_6-METHYLADENOSINE BY ENZYME-ASSISTED ADENOSINE DEAMINATION Article 02 January 2023 BID-SEQ FOR

TRANSCRIPTOME-WIDE QUANTITATIVE SEQUENCING OF MRNA PSEUDOURIDINE AT BASE RESOLUTION Article 15 November 2023 DATA AVAILABILITY Previous published datasets generated or analyzed using the

current protocol are available in the Gene Expression Omnibus repository, under accession no. GSE107838. CODE AVAILABILITY Previous published source code required for analysis has been

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Article  Google Scholar  Download references ACKNOWLEDGEMENTS We thank the members of the laboratory of Z.M. and T. Jaber for helpful discussions. We thank M. Maragkakis for help in

designing custom snRNAs oligonucleotides. This study was technically supported by the Functional Genomics Core at the University of Pennsylvania. This study was financially supported by

grants from the ALS Therapy Alliance (2013-S-014) and the NIH (GM072777) to Z.M. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Division of Neuropathology, Department of Pathology and

Laboratory Medicine, University of Pennsylvania, Philadelphia, PA, USA Fadia Ibrahim & Zissimos Mourelatos * Institute for Translational Medicine and Therapeutics, University of

Pennsylvania, Philadelphia, PA, USA Fadia Ibrahim & Zissimos Mourelatos * Penn Medicine Translational Neuroscience Center, Perelman School of Medicine, University of Pennsylvania,

Philadelphia, PA, USA Fadia Ibrahim & Zissimos Mourelatos Authors * Fadia Ibrahim View author publications You can also search for this author inPubMed Google Scholar * Zissimos

Mourelatos View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS F.I. and Z.M. conceived Akron-seq protocol. F.I. developed, optimized,

performed, and interpreted the Akron-seq results with insightful input from Z.M. F.I. wrote the manuscript with input and editing from Z.M. CORRESPONDING AUTHOR Correspondence to Fadia

Ibrahim. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. ADDITIONAL INFORMATION JOURNAL PEER REVIEW INFORMATION: _Nature Protocols_ thanks Kristian Baker

and other anonymous reviewer(s) for their contribution to the peer review of this work. PUBLISHER’S NOTE: Springer Nature remains neutral with regard to jurisdictional claims in published

maps and institutional affiliations. RELATED LINK KEY REFERENCE USING THIS PROTOCOL Ibrahim, F., Maragkakis, M., Alexiou, P. & Mourelatos, Z. _Nat. Struct. Mol. Biol_. 25, 302–310

(2018): https://doi.org/10.1038/s41594-018-0042-8 SUPPLEMENTARY INFORMATION REPORTING SUMMARY RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Ibrahim,

F., Mourelatos, Z. Capturing 5ʹ and 3ʹ native ends of mRNAs concurrently with Akron sequencing. _Nat Protoc_ 14, 1578–1602 (2019). https://doi.org/10.1038/s41596-019-0151-9 Download citation

* Received: 05 July 2018 * Accepted: 19 February 2019 * Published: 10 April 2019 * Issue Date: May 2019 * DOI: https://doi.org/10.1038/s41596-019-0151-9 SHARE THIS ARTICLE Anyone you share

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