Quantification of basal RNA mutation rate in the repRNA-v4 EGFP region

REPLACE was engineered from an orthogonal alphaviral RNA replication system. It generates a large, continuously diversified library of replicative RNAs through a replicase-limited mode of replication and inducible mutagenesis. We analyzed the mutation frequency at Day0 (IVT RNA), Day1, Day3, Day7, Day14, Day21 and DNA plasmid to estimate the RNA mutation rate. This data was used to guide the construction of RNA mutant libraries. Overall design: EGFP was cloned into the repRNA-v4-derived vector, which was then transcribed in vitro into RNA and delivered into host cells via electroporation. After 24h of electroporation, cells were subjected to continuous puromycin treatment (10 µg/ml) with regular passages.We collected DNA plasmid, IVT RNA and cell samples at Day1, Day3, Day7, Day14, Day21 for sequencing.