¬¬¬¬¬¬¬¬¬¬¬Probing the functional constraints of influenza A virus NEP by deep mutational scanning

Primer extension assay was used to determine transcription-to-replication switch. RNA from infected cells were harvested at the indicated timepoints and transcription-to-replication switch was calculated by using mRNA to cRNA ratio. The switch was defined as the peak of mRNA to cRNA ratio, indicating maximum viral transcription (more mRNA) before transitioning to viral replication (more cRNA). For the primer extension assay, mRNA and cRNA levels were normalized to 5S rRNA as described previously. Raw data files from each three independent biological replicates are included.