Molecular Biology International / 2012 / Article / Fig 6

Review Article

Probing Retroviral and Retrotransposon Genome Structures: The “SHAPE” of Things to Come

Figure 6

Summary of SHAPE-Seq methodology. (a) Input RNAs are bar-coded during in vitro transcription, followed by refolding under desired conditions and modification with SHAPE reagent (NMIA, 1M7). (b) The mixture is split into NMIA-treated and control pools. (c) Reverse transcription is performed with end-labeled primer containing a “handle” at the 5′ end and an Illumina adapter t. (d) The process is followed by hydrolysis of RNA and single-stranded (ss) cDNA ligation to incorporate the second Illumina adapter b. (e) After 9 to 12 cycles of PCR amplification, the cDNA library is analyzed by multiplex paired-end deep sequencing (f) adapted from [32].