In Vivo Click Selective 2ê-Hydroxyl Acylation and Profiling Experiment
icSHAPE provides accurate predictions of RNA-protein interactions and m6A modification in vivo by combining SHAPE-Seq with click chemistry for enhanced isolation (Spitale et al., 2015) (Flynn et al., 2016). , Secondary RNA structures are modified by the addition of a custom 2-methylnicotinic acid imidazolide (NAI) probe, termed NAI-N3. The modifed RNA is marked selectively with dibenzocyclooxtyne (DIBO)-biotin through copper-free click chemistry, enabling purification by streptavidin pull-down.
Briefly, NAI-N3 is added to RNA in vivo to mark it selectively for DIBO-biotin tagging. The cells are lysed, the RNA is poly(A)-selected, tagged with DIBO-biotin, and fragmented. The RNA strands are 3′-end-repaired with T4 PNK and ligated to 3′ adapters. After size-selection, the RNA strands are reverse-transcribed, and both the RNA and first-strand cDNA are captured on streptavidin beads. Another cDNA size-selection step is carried out before circularization and PCR amplification. The samples are ready for NGS.
Similar methods: SHAPE-Seq
- Provides accurate predictions of RNA-protein interactions and m6A modifications in vivo
- Can be applied to ex vivo applications with slight modifications
- Chemical modification is applicable to all nucleotides, unlike DMS which only modifies adenines and cytosines
- Circularization may introduce additional bias
Illumina Library prep and Array Kit Selector
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Flynn R. A., Do B. T., Rubin A. J., et al. 7SK-BAF axis controls pervasive transcription at enhancers. Nat Struct Mol Biol. 2016;
Lu Z., Zhang Q. C., Lee B., et al. RNA Duplex Map in Living Cells Reveals Higher-Order Transcriptome Structure. Cell. 2016;165:1267-1279