Identification of variant distribution in ethanol-selected sRNA libraries

In this study we subjected a library of synthetic sRNAs to multiple rounds of ethanol selection and analyzed changes in sRNA variant abundance by high-throughput sequencing. A synthetic sRNA library was generated by randomizing the first nine nucleotides of a RybB scaffold. The library was put under control of the IPTG-inducible PL promoter on a broad host range plasmid backbone and transferred into V. cholerae ΔrpoE. For laboratory selection experiments, the initial ΔrpoE sRNA library and control strains (WT- pCtr, ΔrpoE-pCtr and ΔrpoE-pRybB) were grown to exponential phase and challenged with ethanol. Following 6h of incubation, cells were recovered on agar plates to test for survival. At least 1 million single clones were pooled to generate the enriched sRNA libraries, which were used as input for the next round of selection following the same protocol. High-throughput sequencing of the isolated plasmids after each selection step was used to determine library complexity and distribution of the sRNA variants.