(A) Schematic of the fluorescent-timer-AI reporter (FT-AI) and of the experimental design. HeLa M2 cells were transfected with a FT-AI reporter. After induction of L1 expression (24 hr, 1 μg/ml doxycycline), cells undergoing retrotransposition are blue and can be sorted by FACS or directly stained with SYTO61 for cell cycle analysis. Within about 2.7 hr of doxycycline treatment 50% of the cells will start to express the FT reporter as estimated in Figure 7—figure supplement 1A. After about 2.35 hr (Figure 7—figure supplement 1B) from expression of the blue FT (retrotransposition event), cells start to become red. Double negative (blue-/red-) cells were also collected by sorting and analyzed as control. The sorted cells are then stained with PI and their cell cycle stage determined. (tet = Tetracycline inducible promoter, 3’=3’UTR, pA = polyA signal, FT = fluorescent timer, SD = splice donor, SA = splice acceptor, CMV = cytomegalovirus constitutive promoter, red round arrow = L1 ‘jumping’). (B) Histogram of cell cycle distribution of sorted and PI stained blue-red- cells (black line) and blue+red- cells (blue histogram). The percentage of cells in each cell cycle stage after sorting and PI staining are reported in Figure 7—figure supplement 2B. (C) Cell cycle analysis using SYTO61 dye of blue- cells that did not undergo retrotransposition (black line), of blue+ cells expressing lower red signal (blue histogram) or blue+ cells expressing higher red signal (purple histogram). The complete profiles of analysis for the reported cells are presented in Figure 7—figure supplement 2C–D.