For M-FISH, chromosome-specific DNA libraries were generated from flow-sorted chromosomes provided by the Flow Cytometry Core Facility of the Wellcome Trust Sanger Institute, using the GenomePlex Complete whole-genome amplification kit (Sigma–Aldrich). A mouse 21-colour painting probe was prepared following the pooling strategy36. Five mouse-chromosome pools were each labelled with ATTO 425-, ATTO 488-, Cy3-, Cy5- and Texas Red-dUTPs (Jena Bioscience), respectively, using the GenomePlex WGA reamplification kit (Sigma–Aldrich) and a dNTP mixture as described previously37. The labelled products were pooled and sonicated to achieve a size range of 200–1,000 bp, optimal for use in chromosome painting. The sonicated DNA was ethanol-precipitated together with mouse Cot-1 DNA (Thermo Fisher Scientific), 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). Bone marrow cells suspended in fixative as described above (3:1 methanol:acetic acid) were dropped onto precleaned microscope 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
