Humancytosnp 12 assay

SNP Chromosome microarray analysis was implemented to confirm genome integrity by examining genomic DNA extracted from hiPSC cells. The Illumina Human CytoSNP-12 assay (Illumina, USA) was performed according to manufacturer's protocols. In summary, 300 ng of hiPSC gDNA underwent whole-genome amplification overnight at 37°C, before fragmentation, purification, and loading onto a Human CytoSNP-12 BeadChip for overnight hybridisation at 48°C. The next day, the BeadChip underwent single base-pair extension and staining using the illumina Automation Control program (Illumina, USA) on the Tecan Freedom EVO liquid handler (Tecan, Switzerland). SNP microarray analysis was performed using BlueFuse Multi v4 (Illumina, USA), with the variant calling algorithm settings as follows: dosage log ratio quality metric (DLRDev) < 0.24, ≥8 consecutive adjacent SNP probes consistent with the copy number change detected, with LogR ratios being > +0.2 and < -0.3 for duplication and deletions, respectively.

Genomic DNA was isolated from the patients’ respective fibroblast and iPSC pellets (passage 10) using the Maxwell® RSC Instrument and Maxwell® RSC Cultured Cells DNA Kit (Promega) according to manufacturer’s protocol. Subsequently, a HumanCytoSNP-12 assay (Illumina) was performed according to the Infinium HD Assay Ultra Automated Protocol using an iScan System (Illumina). The obtained data was analysed in CNV-WebStore, an in-house developed online platform to analyse and interpret microarray data, to investigate the presence of CNVs between the original cell line and the created iPSC clones (Vandeweyer et al., 2011 (link)).