Comparison of RNA quality in fresh and RAID-fixed cells with 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. Overall design: 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 doxycycline-inducible oncogenic HRASG12V were treated with replication stress inducing drugs, gemcitabine + prexasertib (referred to as GP). In this experiment, cells were not treated with doxycycline, and thus have no expression of HRASG12V. Cells in S and G2 phase were selected based on expression of the FUCCI marker Geminin1-110. Cells were either freshly sorted prior to scRNA sequencing (fresh) or they were reversibly fixed, intracelularly stained with yH2AX, and decrosslinked prior to scRNA sequencing (RAID). Fresh cells and RAID-fixed cells were each sorted into 384-well plates, with half of each plate containing veh-treated cells and the other half containing GP-treated cells. After sorting, scRNA sequencing was performed to correlate the degree of yH2AX staining to transcriptional profiles of individual cells.