A38 212

RFe iPSCs were treated with 100 ng/ml Colcemid solution (Life Technologies, 15210040) for 16 hours, then treated with 0.05% trypsin-EDTA for 15 minutes and filtered through a 40 μm cell strainer to remove clumps. RFe cells were collected by centrifugation, resuspended in 1 ml 0.075 M potassium chloride (Sigma-Aldrich, P9327) and incubated for 20 minutes at room temperature. 0.5 ml fixative [1 part glacial acetic (Fisher Scientific, A38-212) mixed with 3 parts methanol (Sigma-Aldrich, A412-4)] were added, RFe cells were collected as before, resuspended in 4 ml fixative, and incubated for 20 minutes at room temperature. The fixation step was repeated, the RFe cells collected as before and all but about 200 μl of the fixative was removed. The cells were resuspended in the remaining fixative and dropped onto slides that were precooled at −20°C. The slides were airdried and the RFe cells stained for 10 minutes with Giemsa Staining solution consisting of 1 part Giemsa solution (Life Technologies, 10092013), and 3 parts Gurr buffer (Invitrogen, 10582013). The slides were washed with water, dried, and mounted in Cytoseal 60 (Thermo Scientific, 23-244257). High-resolution pictures of chromosome spreads were acquired with an AxioObserver microscope (Zeiss) using the 100x oil objective.