Oligopaint libraries were designed as previously described, using the Oligoarray 2.1 software (31) and the hg19 genome build, and purchased from CustomArray. Chromosome paints were designed to have 42 bases of homology with a density of 1.3 probes/kb measured with a 50-kb sliding window. Coordinates for all paints can be found below (Table 1). Oligopaints were synthesized, as previously described (21).

Fluorescence in situ hybridization (FISH) was performed as previously described, with a few modifications to the protocol (21). In short, cells from log-phase cultures were settled on poly-l-lysine–treated glass slides for 30 to 60 min and subsequently fixed for 10 min with 4% formaldehyde in PBS-T [1× phosphate-buffered saline (PBS) with 0.1% Triton X-100] at room temperature, followed by three 5-min washes in PBS-T at room temperature. Slides were then permeabilized by washing with PBS-T 0.3% for 15 min at room temperature. Next, slides were subjected to an ethanol row for drying: 70, 90, and 100% for 5 min each at −20°C. Slides were then allowed to dry for 10 min at room temperature. After drying, slides were rehydrated by washing once for 5 min in 2× SSCT (0.3 M NaCl, 0.03 M sodium citrate, and 0.1% Tween-20) at room temperature, followed by one wash in 2× SSCT/50% formamide for 5 min at room temperature. Slides were then predenatured in 2× SSCT/50% formamide at 92°C for 2.5 min and then in 2× SSCT/50% formamide at 60°C for 20 min. Primary Oligopaint probes in hybridization buffer (10% dextran sulfate/2× SSCT/50% formamide/4% polyvinylsulfonic acid) were then added to the slides, covered with a coverslip, and sealed with rubber cement. Slides were denatured on a heat block in a water bath set to 92°C for 2.5 min, after which slides were transferred to a humidified chamber and incubated overnight at 37°C. Each Oligopaint probe (100 pmol) was used per slide in a final volume of 25 μl. Approximately 16 to 18 hours later, coverslips were removed with a razor blade, and slides were washed in 2× SSCT at 60°C for 15 min, 2× SSCT at room temperature for 15 min and in 0.2× SSC at room temperature for 5 min. Secondary probes (10 pmol per 25 μl) containing fluorophores were added to slides, again resuspended in hybridization buffer, and covered with a coverslip sealed with rubber cement. Slides were incubated at 37°C for 2 hours in a humidified chamber, followed by washes in 2× SSCT at 60°C for 15 min, 2× SSCT at room temperature for 15 min, and 0.2× SSC at room temperature for 5 min. All slides were washed with Hoechst DNA stain (1:10,000 in PBS) for 5 min, followed by two 5-min washes in PBS before mounting in Slowfade (Invitrogen).

Images were acquired on a Leica wide-field fluorescence microscope, using a 1.4–numerical aperture 63× oil-immersion objective (Leica) and Andor iXon Ultra emCCD camera. All images were processed using the Leica LAS-X 3.3 software, deconvolved using Huygens Essential deconvolution software, and saved as leica image file (LIF) files. Images were segmented and measured using a modified version of the TANGO 3D-segmentation plugin for ImageJ (5). Chromosome paints were segmented using a hysteresis-based segmentation algorithm. Statistical tests were performed using Prism 7 software by GraphPad.

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