FIGURE 4 | PARS and Frag-seq methods.

From the following article:

Understanding the transcriptome through RNA structure

Yue Wan, Michael Kertesz, Robert C. Spitale, Eran Segal & Howard Y. Chang

Nature Reviews Genetics 12, 641-655 (September 2011)

doi:10.1038/nrg3049

Understanding the transcriptome through RNA structure

a | Parallel analysis of RNA structure (PARS) strategy. In PARS, poly(A) selected RNA is folded in vitro and incubated with either RNase V1 or S1 nuclease to probe for double- and single-stranded regions, respectively. RNase V1 and S1 nuclease cleave, resulting in a 5′P leaving group. The enzymatically probed RNA is then fragmented. As enzymatic cleavage products contain 5′P, whereas fragmentation and degradation products have 5′OH, only true structure-probing sites can be ligated to adaptors and reverse transcribed. The cDNA library is sequenced using high-throughput sequencing and the resulting reads are mapped to the genome to identify double- or single-stranded regions in the transcriptome. A PARS score can be calculated at each base, whereby a positive PARS score indicates that a base is double-stranded, and a negative PARS score indicates that a base is single-stranded. b | Fragmentation sequencing (Frag-seq) strategy. Nuclear RNA is folded in vitro and probed in solution with P1 endonuclease. P1 cleaves at single-stranded regions, resulting in a 5′P leaving group. This 5′P can be captured by adaptor ligation followed by reverse transcription and high-throughput sequencing. Sequencing reads are mapped back to the genome to identify where single-stranded bases are located in the transcriptome. Frag-seq also contains controls that include sequencing of endogenous 5′P and 5′OH that are originally present in the untreated RNA samples. A cutting score can be calculated at each base that incorporates reads from P1 nuclease and reads from endogenous degradation or fragmentation products. A positive cutting score indicates that the base is single-stranded. Part a is modified, with permission, from Ref. 21 © (2010) Macmillan Publishers Ltd. All rights reserved.

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