SMARTI: Pae Gdm-II riboswitch transcribed with 1.25 mM Gdm (Replicate A)
A mutagenized library of template DNA was made via doped oligos from Keck Oligo Synthesis Resource at Yale University.   Upstream of the aptamer were nonmutagenized regions including the T7 promoter and a handle for downstream PCR steps. This library served as the template for in vitro transcription reactions, and the resulting mutant RNA library was purified via 10% PAGE. The RNA was refolded in the presence of the specified concentration of guanidine and subjected to translation initiation followed by RelE cleavage. T4 PNK (NEB) was used to heal the 2'3' cyclic phosphate generated by RelE cleavage before a pre-adenylated adaptor was added to the 3' end using T4 RNA Ligase II, trunc. K227Q (NEB). This adaptor served as a primer for reverse transcription into DNA using SuperScript IV (Thermo Fisher Scientific). Illumina adaptors were added via a one-step PCR reaction with Phusion HF polymerase (NEB) using the adaptor sequence and 5' nonmutagenized region as handles.