Chromosome Metaphase and FISH Analysis

To analyze metaphase chromosomes, cells in metaphase were harvested using standard procedures. Briefly, cells were swollen in 75 mM KCl (at 37°C), fixed in methanol:acetic acid (3:1) and then dropped onto glass slides. To analyze anaphase and cytokinesis-blocked cells, cells were seeded on poly-L-Lysine slides (Sigma-Aldrich) and fixed in methanol:acetic acid (3:1) for fluorescence in situ hybridization (FISH) analysis. BAC clones were used to prepare FISH probes targeting the regions of interests: RP11-439L14 (GenBank: AC012454.8) for FOLD1, RP11-464P18 (GenBank: AC073105.1) for Chr2Q, RP11-799C6 (GenBank: AC068519.1) for Chr1CCEN, RP11-383P16 (GenBank: AC233288.1) for FRAXA (Supplementary Figure 1A). Probes were labeled using the BioNick labeling system (Thermo Fisher Scientific) or DIG-nick translation mix (Sigma Aldrich). FISH was carried out using standard procedures, as previously published (Bjerregaard et al., 2018 (link)). Slides were mounted using Vectashield mounting medium with DAPI (Vector Labs). Images were captured using an Olympus BX63 microscope. Images were analyzed using CellSens (Olympus) or Fiji/ImageJ software. In the analysis of “bent” shape of human chromosome 2 (Chr2), all of the images were analyzed independently by two researchers.