MS2 mutagenesis library
We designed a custom DNA library containing a barcode, E. coli RNA polymerase (RNAP) promoter, RNAP stall sequence, constant region, and degenerate MS2 hairpin sequence. Degenerate bases were introduced into the MS2 hairpin region using hand-mixed bases containing 88% of the consensus base and 4% of each non-consensus base. This degeneracy ratio was chosen to maximize the total number of variants represented on the RNA array as well as the fractional representation of triple mutants. The RNAP promoter, barcode, and Illumina sequencing primers and adapters were subsequently added by PCR. The sequencing library was then bottlenecked to ensure multiple measurements of each RNA variant. We diluted the MS2 hairpin library to approximately 30.6 fM (~9.2x105 molecules) in 50 µL of the same PCR mix and amplified the library to approximately 30 nM (21 cycles).