Chromosome-specific CO-FISH Assay

The CO-FISH assay was adapted from previous studies [35 (link), 36 (link)]. Briefly, exponentially growing cells were cultured overnight in the presence of 10 μM BrdU:BrdC (3:1) (Sigma-Aldrich) at 37 °C to allow for one round of replication. Colcemid (Roche) was then added at a concentration of 0.1 μg/ml for 4 h to arrest cells at prometaphase. After fixation of cellular preparations on slides and Hoechst 33258 (Sigma-Aldrich) staining, the newly synthesized strands were degraded following UV light exposure and treatment with 10 U/μl ExoIII (Promega). Hybridization was performed using fluorescent centromeric PNA probes against CENP-B box motif sequences. The PNA probe labeled with Cy3 (ATTCGTTGGAAACGGGA; PNABio Inc) hybridizes with the leading strand and the reverse PNA probe labeled with Alexa-488 (TCCCGTTTCCAACGAAT; Eurogentec) hybridizes with the lagging strand. DNA was counterstained with DAPI (Sigma-Aldrich). Metaphases were captured on a confocal laser microscope (FV1000 Olympus) and images were analyzed using Image J software (NIH). Quantitation to measure for SCE between α satellite sequences (C-SCE) was done by counting the number of CO-FISH signals showing C-SCE over the total number of CO-FISH signals observed for each metaphase.