Multigenerational cell tracking of DNA replication and heritable DNA damage

The goal of this project was to analyze gene expression changes upon polyploidization. To this end, human U-2 OS cells were exposed to the NEDD8-activating enzyme (NAE) inhibitor Pevonedistat (MLN4924), a first-in-class anticancer drug that induces DNA re-replication, or to oncogenic H-RAS V12 (HRAS) over-expression. Untreated U-2 OS cells served as control. Cells with 2N-4N DNA content were separated from cells with >4N DNA content by FACS and single cells were sorted into 384 well plates for scRNA-seq analysis. For SORT-Seq, cells were sorted on a FACS Aria III 3L system equipped with a 488 nm laser line. Pevonedistat-treated cells were treated for 24 h with 175 nM Pevonedistat. Prior to sorting, cells were incubated for 1 h with a final concentration of 5 µg/ml Hoechst 33342 (Thermo Fisher). Cells were sorted into 384-well plates that were acquired from Single Cell Discoveries. Cells with increased ploidy were gated according to their Hoechst signal and as a reference for the whole population the gate was adjusted in the same sample. Note: The processed files "barcodes.tsv", "features.tsv" and "matrix.mtx" are poisson-corrected data from the raw count tables.