Cells were grown up to 60% confluency after plating. Colcemid (Invitrogen) was added to the cultural medium at a concentration of 100 ng/ml, and cells were incubated at 37°C for 3 to 4 hours. Cells were harvested using trypsin and resuspend in 1 ml of culture medium after spinning down. Five microliters of 37°C prewarmed KCI was added slowly to the cell suspension and incubated at room temperature for 7 to 10 min followed by adding 120 μl of freshly prepared fixative solution (methanol:acetic acid in 3:1 volume ratio). Cells were incubated in 9.5 ml of fixative solution for 10 min after being spun down at 1000 rpm for 8 min and having discarded the supernatant. Cells were then resuspended in 0.3 ml of fixative and dropped on a glass slide before being placed onto slide warmer at 65°C for 20 min followed by treatment with RNAse A (1 mg/ml; 1:100 from Qiagen) and propidium iodide (1 mg/ml stock and 1:1000 final) in 2× SSC for ~45 min at 37°C. Slides were air dried before mounting using mounting medium with DAPI (Vectashield). Chromosome spreads were imaged with 63× oil objective mounted on a Ti-E microscope (Nikon) and analyzed using previously established software (44, 45).

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