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The genomic instability of stem cells in culture, caused by their routine in vitro propagation or by their genetic manipulation, is deleterious both for their clinical application and for their use in basic research. Frequent evaluation of the genomic integrity of stem cells is thus required, and it is usually performed using cytogenetic or DNA-based methods at variable sensitivities, resolutions and costs. Here we present a detailed protocol for determining the genomic integrity of pluripotent stem cells (PSCs) using their global gene expression profiles. This expression-based karyotyping (e-karyotyping) protocol uses gene expression microarray data (either originally generated or derived from the literature) and describes how to organize it properly, subject it to two complementary bioinformatic analyses and conservatively interpret the results in order to generate an accurate estimation of the chromosomal aberrations in the …