Biotin n6 ddatp

After germination of heterozygous rscΔ strains, the ploidy of each strain was monitored by flow cytometry (Fig. 1 b). Genomic DNA from the sample that contained >95% diploid cells with the lowest number of generations was isolated. Genomic DNA from wild-type cells arrested in G₁ with α-factor was used as the control sample. CGH was performed essentially as described previously (Dion and Brown, 2009 (link); Chambers et al., 2012 (link)). In brief, genomic DNA was amplified by using a WGA2 kit (Sigma) and treated with DNaseI (NEB) to fragment DNA to a mean 50-bp length. Then, DNA fragments were labeled with biotin-N⁶-ddATP (Enzo Life Sciences) by using terminal deoxynucleotidyl transferase (Fermentas), hybridized to an S. cerevisiae Tiling Array (Affymetrix), and visualized using streptavidin R-phycoerythrin conjugate (Invitrogen) and normal goat IgG (Sigma). Experiment signal intensities from each microarray were compared with the control sample using Tiling Analysis Software (Affymetrix) using quantile normalization, perfect match probes only, a bandwidth of 60, maximum gap of 80, and minimum run of 40. The CGH profiles were generated with IGB 6.3 (Affymetrix).