Osimertinib

Watermelon cells were thawed, expended in dox (2μg/ml) containing media for 96 hours and mCherry positive cells were sorted using a MoFlo Astrios Cell Sorter (Beckman Coulter) and re-plated. Following 96 hours of recovery, the cells were seeded into six-well plates at 300,000 cell per well and were given 24h to attach prior to adding 300nM osimertinib. Dox was continuously added to the media until day 3 of drug treatment. Cells were harvested at day 0 (untreated), 3, 7 and 14 of drug treatment. To obtain cell suspension for single cell profiling, cells were scrapped from the well, washed and resuspended in FACS buffer (0.5% BSA in phosphate-buffered saline), and filtered through a 40μm strainer. To delineate the differences between persister populations, day 14 cells were gated based on mCherry expression. Following sorting, the cells were spun down and approximately 9,000 single cells per sample were loaded to the Chromium Controller (10x Genomics). ScRNA-seq libraries were generated using the 10X Genomics Chromium Single Cell 3’ Kit v2 and the 10x Chromium Controller (10x Genomics) according to the standard v2 protocol. The resulting 3’ scRNA-Seq libraries were pooled together and sequenced with a HiSeq (Illumina, R2 read length 98 base pairs). To increase lineage barcode capture, targeted sequencing of the barcode area was performed using the whole transcriptome amplification product generated as a part the v2 protocol as a PCR template (for primer list see Supplementary Table 4). Targeted libraries were gel purified and sequenced with a MiSeq (Illumina).