NET-Seq
Structure-Seq/DMS-Seq
Structure-Seq profiles RNA structures for in vivo or in vitro applications with single-nucleotide resolution. This method identifies secondary RNA structures by using the chemical modification induced by DMS on unpaired adenines and cytosines.
Briefly, samples are treated with DMS to mark secondary RNA structures in vivo. The RNA is extracted, poly(A)-selected, and treated with DNase. Using random hexamers as primers, reverse transcription is initiated. The resulting single-stranded DNA is ligated to single-stranded DNA linkers and self-circularized using CircLigase enzyme. Next, the DNA is PCR-amplified, size-selected, and sequenced.
Similar methods: icSHAPE, Mod-Seq, DMS-seq, PARS, Frag-seq, dsRNA-Seq
Pros:
- Provides genome-wide profiling of RNA structures at single-nucleotide resolution
- Can be used for in vivo and in vitro applications
- DMS is cell-permeable and can be used for in vivo applications
- One RT primer synthesis retrieves information for tens of thousands of RNA structures
- Random-hexamer primers minimize 3' end bias
Cons:
- RBPs can block DMS modification in vivo
- Circularization may introduce additional bias
- Structure-Seq: Ding Y., Tang Y., Kwok C. K., Zhang Y., Bevilacqua P. C. and Assmann S. M. In vivo genome-wide profiling of RNA secondary structure reveals novel regulatory features. Nature. 2014;505:696-700
- Protocol: Ding Y., Kwok C. K., Tang Y., Bevilacqua P. C. and Assmann S. M. Genome-wide profiling of in vivo RNA structure at single-nucleotide resolution using structure-seq. Nat Protoc. 2015;10:1050-1066