ZTP Riboswitch RndP3Ext Library combinatorial mutagenesis with 1mM ZMP, Replicate 2
DNA templates consisted of an E. coli promoter followed by DNA encoding the C. beijerinckii pfl Riboswitch with randomized nucleotides at defined positions and a terminal biotin. DNA template was incubated with streptavidin after creating open complexes with E. coli RNA polymerase, followed by initiating in vitro transcription with the addition of Mg++. Reactions were run for 30s at 37C in standard transcription buffer. Extracted RNAs were then processed according a modified version of the SHAPE-Seq v2.1 protocol for cDNA synthesis and library preparation in which Superscript IV Reverse Transcriptase was used in place of Superscript III. (doi:10.1016/j.ymeth.2016.04.002).