Plasmid encoding the parental B. subtilis glmS ribozyme was isolated from DH5-alpha cells using Monarch Plasmid Miniprep Kit (NEB). The methylated plasmid was used to construct a plasmid variant library using nicking mutagenesis (Wrenbeck et al., 2016). The library was in vitro transcribed, allowed to self-cleave at 6 timepoints, purified by PAGE, and reverse transcribed into 12 barcoded (cleaved and uncleaved products for each of the 6 timepoints) output cDNA sub-libraries. The sub-libraries were pooled and sequenced in parallel.