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 fragmentation, cDNA generation, PCR amplification, and deep sequencing. The step-by-step protocol we describe here is optimized for cultured human cells but can be adapted to primary cells and tissues. Akron-seq can be completed within 6 d, and sequencing and analysis can be completed within 6 d.
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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 deposited on GitHub (https://github.com/mnsmar/ribothrypsis).
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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.
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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.
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Journal peer review information: Nature Protocols thanks Kristian Baker and other anonymous reviewer(s) for their contribution to the peer review of this work.
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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
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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
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DOI: https://doi.org/10.1038/s41596-019-0151-9
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