| In Vitro |
The bifunctional NAI-N3 SHAPE reagent is used to label all RNAs inside living cells (or in vitro refolded RNA) to generate covalent modifications on targeted RNAs at single-nucleotide resolution. RNA is isolated, biotinylated using click chemistry and randomly fragmented to a size of 50–100 nt. To generate cDNA sequencing libraries, the RNA fragments are subjected to 3′-end repair, adapter ligation, size selection, biotin enrichment, reverse transcription (RT) and PCR. The end result is selective amplification of fragments corresponding to modified bases in the NAI-N3–treated samples or background expression values from mock-treated samples. Sequencing reads generated from the NAI-N3– and mock (DMSO)-treated samples are mapped to a transcriptome annotation, followed by computational identification of RT stops corresponding to SHAPE modifications—and hence reactive nucleotides—across the entire transcriptome[1].
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