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Scheme of the time series experiment performed on the barcoded MDAMB468 cell line. D0 are untreated parental MDAMB468 cells (MDAMB468-P) a few hours before afatinib addition, and D3 through D40 is the duration of the experiment where cells where subjected to incremental doses of afatinib ranging from 250 to 2000 nM. Cells at D40 were considered as afatinib tolerant persisted cells (ATPC). 10x Chromium sequencing was performed at Day 0 (D0), Day 3 (D3), Day 6 (D6) and Day 9 (D9) of 250 nM afatinib treatment. Quantseq-Flex to retrieve afatinib-tolerant lineages in bulk was performed at Day 40 with 2000 nM afatinib treatment.

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