RNA structures regulate norovirus life cycle and enable rational attenuation in vivo

Two biological replicates were collected for each probing method. For each replicate, one sample is treated with the probing reagent (2A3 for SHAPE-MaP, DMS for fbDMS-MaP; modified), and one sample is treated with a control reagent (DMSO for SHAPE-MaP, ethanol for fbDMS-MaP; unmodified). 2A3 reacts with the RNA backbone of flexible nucleobases, whereas DMS reacts with the Watson-Crick face of unpaired bases.

Both reagents covalently modify the RNA. These modifications are encoded as mutations during reverse transcription with MarathonRT. Amplicons are then PCR amplified, next-generation sequencing libraries are prepared, and sequenced using an Illumina NextSeq 2000 platform.