Chemical Inference of RNA Structures
CIRS-Seq was developed to investigate the complexity of secondary RNA structures in the mammalian transcriptome (Incarnato et al., 2014). CIRS-Seq uses DMS to methylate the N1 of adenosine and N3 of cytosine residues, and CMC to modify pseudouridines selectively, but only when they are in single-stranded conformation. Modifications on these nucleotide residues halt the RT process, effectively producing a truncated cDNA as a marker for the location of secondary RNA structures.
Briefly, cells are lysed and treated with proteinase K to dissociate protein-bound RNAs while leaving RNA secondary structures intact. The lysates are separated into 3 different treatment linesãDMS, CMC, and no treatment. In all 3 treatment lines, total RNA is extracted and reverse-transcribed using random primers. The resulting cDNA is isolated, ligated to sequencing adapters, and subjected to high-throughput sequencing. Reads from the DMS and CMC lines are used to identify the locations of secondary RNA structures, while the control is used to reduce background noise.
- Accurately predicts secondary RNA structures, and reveals features of mRNAs and ncRNAs
- Provides single-base resolution
- Can identify structural requirements for RBPs
- CMC and DMS may react with non_secondary RNA structures
Illumina Library prep and Array Kit Selector
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Incarnato D., Anselmi F., Morandi E., et al. High-throughput single-base resolution mapping of RNA 2′-O-methylated residues. Nucleic Acids Res. 2016;