Optimizing scheduling in dual-pulse nucleoside labeling experiments for cell-cycle analysis

All eukaryotic cells go through a universal sequence of phases during their division cycle, where the phase timings vary according to cell type and state. Dual-pulse nucleoside labeling (DPNL) is a standard, widely applicable experimental DNA base-substituting technique to probe cell-cycle kinetics at the population level, including in living organisms. In such an experimental protocol, a key scheduling parameter is the choice of waiting time between the two labeling pulses. Here, we model population cell-cycle dynamics as a three-stage Poisson process with an idealized S-phase labeling step and use a simulation-based look-up procedure to demonstrate that the inter-pulse waiting time can be optimized to maximize the signal-to-noise ratio of inferred cycle parameters-an issue that is especially critical in DPNL experiments with limited cell numbers and replicates. An optimal choice of pulse scheduling typically improves S-phase time inference by 50% compared to a suboptimal choice. We further discuss the procedure to perform such a task in an experimentally relevant setting.