Identification of heterogenous expressed transcripts that mitigate drug-induced replication stress

The underlying mechanisms that influence cancer cells' sensitivity to replication stress are impeded by the analysis of bulk-samples which neglect tumor heterogeneity and fail to accurately interpret cell cycle mediated resistance. Here, by combining intracellular immunostaining and RNA-sequencing of single cells, we characterized the transcriptomes of subsets of cells with oncogenic RAS that exhibit low levels of RS even when challenged with a CHK1 inhibitor and gemcitabine. All samples consist of human RPE cell lines harboring the Tet Repressor, FUCCI4 system, fluorescently tagged 53BP1 and HRASG12V, which are described in Segeren et al 2022. Cells with WT HRAS and with doxycycline-inducible oncogenic HRASG12V were treated with replication stress inducing drugs, gemcitabine + prexasertib (referred to as GP). Single cells cells were sorted based on their degree of DNA damage as determined by the level of intracellular yH2AX staining. S-phase cells only were selected based on the level of DAPI staining. After sorting, scRNA sequencing was performed to correlate the degree of yH2AX staining to transcriptional profiles of individual cells. One 384-well plate was sorted for the Veh and Dox treated cells that were not treated with gemcitabine/prexasertib, and 3 384-well plates were sorted for the Veh and dox treated cells that were treated with gemcitabine/prexasertib.