DNA Fiber Assay with Fluorescent Labeling

DNA fiber assay was performed as described previously with some modifications (Schwab et al., 2015 (link)). In brief, exponentially growing cells were first incubated with 25 μM iododeoxyuridine (IdU) and then with 125 μM chlorodeoxyuridine (CldU) for the indicated times. Fiber spreads were prepared from 0.5 x10⁶ cells/ml. Slides were stained as described previously . A confocal microscope (LSM 510 Meta or LSM 710 Meta; Carl Zeiss) equipped with Plan-Apochromat 63 × /1.4 oil DIC objective was used to collect fiber images from randomly selected fields at RT using ZEN 2009 software (Carl Zeiss). Analysis was performed using the ImageJ software package (National Institutes of Health). A minimum of 100 fibers or 20 sister fork pairs per experiment from at least three independent experiments was scored. Mann-Whitney test was used to determine statistical significance. On boxplots whiskers indicate 5-95 percentile.