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Chemical mapping methods probe RNA structure by revealing and leveraging correlations of a nucleotides 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.
Chemical mapping is a widespread technique for structural analysis of nucleic acids in which a molecules reactivity to different probes is quantified at single-nucleotide resolution and used to constrain structural modeling. This experimental framewo
Next-generation RNA sequencing (RNA-seq) technology has been widely used to assess full-length RNA isoform abundance in a high-throughput manner. RNA-seq data offer insight into gene expression levels and transcriptome structures, enabling us to bett
Background: High-throughput techniques bring novel tools but also statistical challenges to genomic research. Identifying genes with differential expression between different species is an effective way to discover evolutionarily conserved transcript
We have established an RNA Mapping Database (RMDB) to enable a new generation of structural, thermodynamic, and kinetic studies from quantitative single-nucleotide-resolution RNA structure mapping (freely available at http://rmdb.stanford.edu). Chemi
Chemical purity of RNA samples is critical for high-precision studies of RNA folding and catalytic behavior, but such purity may be compromised by photodamage accrued during ultraviolet (UV) visualization of gel-purified samples. Here, we quantitativ