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RNA Folding pp 95–117Cite as

Massively Parallel RNA Chemical Mapping with a Reduced Bias MAP-Seq Protocol

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1086))

Abstract

Chemical mapping methods probe RNA structure by revealing and leveraging correlations of a nucleotide’s structural accessibility or flexibility with its reactivity to various chemical probes. Pioneering work by Lucks and colleagues has expanded this method to probe hundreds of molecules at once on an Illumina sequencing platform, obviating the use of slab gels or capillary electrophoresis on one molecule at a time. Here, we describe optimizations to this method from our lab, resulting in the MAP-seq protocol (Multiplexed Accessibility Probing read out through sequencing), version 1.0. The protocol permits the quantitative probing of thousands of RNAs at once, by several chemical modification reagents, on the time scale of a day using a tabletop Illumina machine. This method and a software package MAPseeker (http://simtk.org/home/map_seeker) address several potential sources of bias, by eliminating PCR steps, improving ligation efficiencies of ssDNA adapters, and avoiding problematic heuristics in prior algorithms. We hope that the step-by-step description of MAP-seq 1.0 will help other RNA mapping laboratories to transition from electrophoretic to next-generation sequencing methods and to further reduce the turnaround time and any remaining biases of the protocol.

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Acknowledgements

We thank members of the Das laboratory for discussions and tests of the protocol. We thank Tom Mann and Frank Cochran for preparing the 1M7 acylating reagent, and S. Mortimer for an updated 1M7 synthesis protocol. Writing was supported by the NIH (IRACDA fellowship to MS; R01GM100953 to RD) and the Burroughs-Wellcome Foundation (Career Award to RD).

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Authors and Affiliations

  1. Department of Biochemistry, Stanford University, Stanford, CA, USA

    Matthew G. Seetin, Wipapat Kladwang, John P. Bida & Rhiju Das

  2. Department of Physics, Stanford University, Stanford, CA, USA

    Rhiju Das

Authors
  1. Matthew G. Seetin
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  2. Wipapat Kladwang
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  3. John P. Bida
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  4. Rhiju Das
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Editor information

Editors and Affiliations

  1. Dept. of Biochemistry & Cell Biology, University of Vienna Max F. Perutz Laboratories, Vienna, Austria

    Christina Waldsich

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Seetin, M.G., Kladwang, W., Bida, J.P., Das, R. (2014). Massively Parallel RNA Chemical Mapping with a Reduced Bias MAP-Seq Protocol. In: Waldsich, C. (eds) RNA Folding. Methods in Molecular Biology, vol 1086. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-667-2_6

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  • DOI: https://doi.org/10.1007/978-1-62703-667-2_6

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-666-5

  • Online ISBN: 978-1-62703-667-2

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