Selection of activating EGFR mutations from randomly mutated EGFR libraries. Selection for ligand-independent EGFR activation

In this study, a randomly mutated human EGFR library (exons only) was selected for ligand-independent EGFR-activation in HEK293T cells using a high-throughput assay termed PhosphoFlowSeq. Briefly, HEK293T cells were transfected with plasmids containing randomly mutated EGFR genes. Next, the HEK293T cells expressing the mutated EGFR variants were screened for EGFR-phosphorylation in the absence of EGFR ligands using flow cytometry, followed by plasmid isolation from enriched cells, PCR-amplification of EGFR genes and deep sequencing analysis (Illumina sequencing).