180 k cgh arrays

aCGH was performed on DLD-1 cell lines (2N, 4N, PTA, and trisomic clones) as previously described (Ruiz et al., 2011 (link); Juskevicius et al., 2016 (link)), with minor modifications. PTA clones were analyzed two passages after establishing the lines. In brief, 1 μg of sample DNA and equal amounts of female reference genomic DNA (Promega 46/XX, Madison, WI) were digested with DNaseI to a size range of 200–500 base pairs. Subsequent labeling of sample and reference DNA with Cy3-dUTP and Cy5-dUTP, respectively, was performed with the BioPrime Array CGH Genomic Labeling System (Invitrogen, Carlsbad, CA). Labeling efficiency was quantified by measuring the specific activity of the incorporated dyes with a Nanodrop (Thermo Fischer Scientific, Waltham, MA). Reference and sample DNA were mixed and hybridized to 180k CGH arrays (Agilent Technologies, Santa Clara, CA) for 24 h in a rotating oven at 67°C. Microarray slides were scanned with the Agilent 2565C DNA scanner and images were analyzed with Agilent’s Feature Extraction using default settings. Feature extracted array CGH data were evaluated using Agilent’s CytoGenomics software v3.0.1.1. Aberrations were called with the aberration detection algorithm ADM2 set to a threshold of 12.0, with Fuzzy Zero and GC-content (window size: 2kb) correction. A minimum of three probes was necessary to call an aberration.