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Related Concept Videos

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Massively parallel RNA chemical mapping with a reduced bias MAP-seq protocol.

Matthew G Seetin1, Wipapat Kladwang, John P Bida

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

Methods in Molecular Biology (Clifton, N.J.)
|October 19, 2013
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Summary

Multiplexed Accessibility Probing read out through sequencing (MAP-seq) enables quantitative RNA structure probing for thousands of molecules daily. This optimized protocol and software (MAPseeker) reduce bias and transition labs to next-generation sequencing methods.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Genomics

Background:

  • Chemical mapping methods reveal RNA structure by correlating nucleotide accessibility/flexibility with chemical probe reactivity.
  • Previous methods were limited in throughput and required extensive analysis like slab gels or capillary electrophoresis.
  • Pioneering work enabled probing hundreds of molecules using Illumina sequencing, but optimizations were needed.

Purpose of the Study:

  • To describe optimizations to chemical mapping methods for RNA structure probing.
  • To introduce the Multiplexed Accessibility Probing read out through sequencing (MAP-seq) protocol, version 1.0.
  • To provide a method and software (MAPseeker) for quantitative, high-throughput RNA structure analysis.

Main Methods:

  • Development and optimization of the MAP-seq protocol for quantitative RNA probing.
  • Utilizing a tabletop Illumina sequencing machine for high-throughput analysis.
  • Implementing the MAPseeker software package to address biases and improve analysis.

Main Results:

  • MAP-seq enables quantitative probing of thousands of RNAs simultaneously using chemical modification reagents.
  • The protocol operates on the timescale of a day using standard Illumina sequencing.
  • MAPseeker software eliminates PCR steps, improves adapter ligation, and avoids problematic algorithmic heuristics, reducing bias.

Conclusions:

  • MAP-seq 1.0 offers a streamlined, quantitative, and high-throughput method for RNA structure probing.
  • The protocol facilitates the transition from traditional electrophoretic methods to next-generation sequencing for RNA mapping.
  • Further reductions in turnaround time and remaining biases are anticipated with this optimized protocol and software.