Sister chromatid cohesion was analyzed in metaphase spreads in all nine HeH ALL patient samples subjected to scWGS. The percentage of cells with cohesion defects, measured as visible primary constriction gaps (gaps between the sister chromatids at the centromeres)14 (link), was counted. FISH metaphase spreads mounted with DAPI were used for the assay, where 20–39 cells were analyzed per case. Images were captured using a Z2 fluorescence microscope (Zeiss, Germany) and the CytoVision software (v7.4, Leica, Germany).
Metaphase FISH was carried out on cases 2, 3, and 4 according to standard methods, with a total of 17–35 cells captured for each analysis. All whole chromosome paint FISH probes were acquired from Applied Spectral Imaging (Carlsbad, CA), and locus-specific probes from Vysis (Abbot Laboratories, Chicago, IL). FISH analysis was performed as follows: slides from case 2 were hybridized with whole chromosome paint probes for chromosomes 1 (Aqua – blue), 6 (Cy3 – red), and 21 (FITC – green); for case 3, whole chromosome paint probes for chromosomes 1 (FITC) and 16 (Aqua) were used together with a telomeric probe for 16q (Cy3); and for case 4, one analysis was performed with whole chromosome paint probes for chromosomes 3 (FITC) and 6 (Cy3), and another analysis for chromosome 14 (Aqua) together with a LSI TRA/D (14q11.2) break-apart dual colour probe (Cy3/FITC).
Metaphase FISH was carried out on cases 2, 3, and 4 according to standard methods, with a total of 17–35 cells captured for each analysis. All whole chromosome paint FISH probes were acquired from Applied Spectral Imaging (Carlsbad, CA), and locus-specific probes from Vysis (Abbot Laboratories, Chicago, IL). FISH analysis was performed as follows: slides from case 2 were hybridized with whole chromosome paint probes for chromosomes 1 (Aqua – blue), 6 (Cy3 – red), and 21 (FITC – green); for case 3, whole chromosome paint probes for chromosomes 1 (FITC) and 16 (Aqua) were used together with a telomeric probe for 16q (Cy3); and for case 4, one analysis was performed with whole chromosome paint probes for chromosomes 3 (FITC) and 6 (Cy3), and another analysis for chromosome 14 (Aqua) together with a LSI TRA/D (14q11.2) break-apart dual colour probe (Cy3/FITC).
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