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

TL;DR

The paper introduces MAP-seq 1.0, an optimized, high-throughput RNA chemical mapping protocol that reduces bias and enables rapid, multiplexed analysis of thousands of RNAs using next-generation sequencing.

Contribution

The authors present an improved MAP-seq protocol that eliminates PCR bias, enhances ligation efficiency, and streamlines RNA structure probing on Illumina platforms.

Findings

Allows probing of thousands of RNAs in a day

Reduces bias by eliminating PCR steps

Improves ligation efficiency of adapters

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 table-top 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.