Acetone

Metaphase chromosomes from hIPSCs were harvested as described previously (Moralli et al., 2011 (link)), with slight modifications. Briefly, after refreshing, the medium cells were treated with 10 μM ROCK inhibitor (Tocris) for 2–3 hr, followed by incubation with 25–100 ng/ml Nocodazole (Sigma-Aldrich) for a further 2–3 hr. Subsequently, cells were dissociated using Accutase (Sigma-Aldrich) and treated with a buffered hypotonic solution (0.4% KCl in 10 mM HEPES [pH 7.4]) for 8–12 min at 37°C. The cells were then fixed and washed twice in a 6:1 (v/v) methanol:glacial fixative and kept in fixative in −20°C freezer until use.
For multiplex-fluorescence in situ hybridization (M-FISH), chromosome-specific DNA libraries were generated from 5,000 copies of flow-sorted chromosomes, provided by Flow Cytometry Core Facility of the Wellcome Trust Sanger Institute, using GenomePlex Whole Genome Amplification (WGA2) kit (Sigma-Aldrich). Human 24-color painting probe was made following the pooling strategy (Geigl et al., 2006 (link)). Five human chromosome pools were labeled with ATTO 425-, ATTO 488-, CY3-, CY5-, and Texas Red-dUTPs (Jena Bioscience), respectively, using WGA 3 re-amplification kit (Sigma-Aldrich) as described before (Gribble et al., 2013 (link)). The labeled products were pooled and sonicated to achieve a size range of 200–1,000 bp, optimal for use in chromosome painting. Then the sonicated DNA sample was precipitated with ethanol together with human Cot-1 DNA (Invitrogen) and resuspended in a hybridization buffer (50% formamide, 2 × SSC, 10% dextran sulfate, 0.5 M phosphate buffer, 1 × Denhardt’s solution [pH 7.4]). Metaphase preparations were dropped onto precleaned microscopic slides followed by fixation in acetone (Sigma-Aldrich) for 10 min and dehydration through an ethanol series (70%, 90%, and 100%). Metaphase spreads on slides were denatured by immersion in an alkaline denaturation solution (0.5 M NaOH, 1.0 M NaCl) for 50–60 s, followed by rinsing in 1M Tris-HCl (pH 7.4) solution for 3 min, 1 × PBS for 3 min, and dehydration through a 70%, 90%, and 100% ethanol series. The M-FISH probe was denatured at 65°C for 10 min before being applied onto the denatured slides. The hybridization area was sealed with a 22 × 22-mm coverslip and rubber cement. Hybridization was carried out in a 37°C incubator for 2 nights. The posthybridization washes included a 5-min stringent wash in 0.5 × SSC at 75°C, followed by a 5-min rinse in 2 × SSC containing 0.05% Tween20 (VWR) and a 2-min rinse in 1 × PBS, both at room temperature. Finally, slides were mounted with SlowFade Gold mounting solution containing 4′6-diamidino-2-phenylindole (Invitrogen). Images were visualized on a Zeiss AxioImager D1 fluorescent microscope equipped with narrow band-pass filters for DAPI, DEAC, FITC, CY3, TEXAS RED, and CY5 fluorescence and an ORCA-EA CCD camera (Hamamatsu). M-FISH digital images were captured using the SmartCapture software (Digital Scientific) and processed using the SmartType Karyotyper software (Digital Scientific). Approximately 10–20 metaphase chromosomes per IPSC line were fully karyotyped.