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. 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.