Oligonucleotide Preparation for REPSA

Single-stranded oligonucleotides used in this study (Supplementary Table S3) were obtained from Integrated DNA Technologies (Coralville, IA). ST2R24 library DNA used for the initial REPSA round was PCR amplified with primers ST2L and IRD7_ST2R for seven cycles to ensure maximal double-stranded DNA content with fully annealed randomized cassette regions. Subsequent REPSA round DNAs were PCR amplified for 6, 9, and 12 cycles to identify those products with optimal cassette integrity. Libraries for massively parallel semiconductor sequencing were prepared by a two-step fusion PCR process, using primers A_BC01_ST2R and trP1_ST2L as the initial set and A_uni and trP1_uni as the second set, as previously described [8 (link)]. Other duplex DNAs were prepared by conventional PCR amplification following the Taq DNA polymerase manufacturer’s instructions. EMSA probes were amplified with primers ST2L and IRD7_ST2R, while nucleic acids used in BLI assays were amplified with primers ST2L and Bio_ST2R. The concentrations for the modified oligonucleotides were measured with Qubit 3 Fluorometer following our protocol [37 (link)].