NET-Seq

icSHAPE

icSHAPE provides accurate predictions of RNA-protein interactions and m6A modification in vivo by combining SHAPE-Seq with click chemistry for enhanced isolation. , 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

Pros:

• 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

Cons:

• Circularization may introduce additional bias

• icSHAPE: Spitale R. C., Flynn R. A., Zhang Q. C., et al. Structural imprints in vivo decode RNA regulatory mechanisms. Nature. 2015;519:486-490
• icSHAPE Protocol: Flynn R. A., Zhang Q. C., Spitale R. C., Lee B., Mumbach M. R. and Chang H. Y. Transcriptome-wide interrogation of RNA secondary structure in living cells with icSHAPE. Nat Protoc. 2016;11:273-290

1. Flynn R. A., Do B. T., Rubin A. J., et al. 7SK-BAF axis controls pervasive transcription at enhancers. Nat Struct Mol Biol. 2016;
2. 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