Yeast chromosome integrity was analyzed as described previously [168 (link)] with certain modifications. Yeast cells grown at 23°C were embedded in 20-μl plugs of low-melting-point SeaKem Gold agarose (Lonza, Basel, Switzerland). The plugs were digested with Zymolyase 100T (BioShop) overnight at 37°C with gentle rotation and then with proteinase K (A&A Biotechnology) and RNase A (Sigma-Aldrich, St. Louis, MO, USA) overnight at 37°C with gentle rotation. Then, the plugs were treated with 2 U or 5 U of S1 nuclease (Thermo Scientific) for 30 min in S1 buffer provided by the manufacturer. Next, plugs were placed in the wells of a 1% D5 agarose gel (BioMaxima, Lublin, Poland) in 1x TAE and sealed with the same agarose. Electrophoresis was performed on a CHEF Mapper XA pulsed-field electrophoresis system (Bio-Rad, Hercules, CA, USA) for 18 h in 1x TAE buffer at 6 V/cm and 12°C, angle of 120°, and switch time of 60–85 s, with a ramp-up of 0.8. After electrophoresis, the DNA was stained with 0.5 μg/ml ethidium bromide (Sigma-Aldrich) and visualized using UV light.
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