Sureprint g3 human cgh microarray kit 8 60k

aCGH was performed using the SurePrint G3 Human CGH Microarray Kit (8×60K) (Agilent Technologies, Santa Clara, CA USA), according to the manufacturer’s recommendations. Labeling and hybridization of patient and reference DNA (#5190-3796, Human Reference DNA, Agilent Technologies, Santa Clara, CA USA) were performed using enzymatic labeling and hybridization protocols (v. 7.5 Agilent Technologies, Santa Clara, CA USA). Array images were acquired with an Agilent SureScan Microarray Scanner (Agilent Technologies, Santa Clara, CA USA). Data analysis was performed using Cytogenomics Software (v. 3.0.6.6) (Agilent Technologies), the publicly available Database of Genomic Variants (DGV), and the Database of Chromosomal Imbalance and Phenotype in Humans employing Ensembl Resources (DECIPHER). Annotations of the genes located within the region of genomic imbalance were retrieved from the NCBI Gene Database, OMIM, and the literature.

Array CGH was performed using the SurePrint G3 Human CGH Microarray Kit 8 × 60 k (Agilent Technologies Inc., Palo Alto, CA, USA). Labeling and hybridization were performed using the SureTag DNA Labeling kit and Oligo aCGH/ChiP‑on‑chip Hybridization kit (Agilent Technologies Inc.), according to the manufacturer’s protocol (Protocol v8.0). Genomic DNA derived from each cell line without acid or DCA was compared to a Human Male Reference DNA (Agilent). Genomic DNA of each cell line without acid or DCA was labeled with Cy5-dUTP and compared to a reference DNA, which was labeled with Cy3-dUTP (i.e., naïve CP-A vs reference). Genomic DNA of each cell line treated with acid and DCA was labeled with Cy5-dUTP and compared to that of corresponding cell line treated without acid or DCA, which was labeled with Cy3-dUTP (i.e., CP-A treated with acid and DCA vs naïve CP-A). After washing the array slides, they were scanned using an Agilent Technologies Microarray scanner, and the resulting data were analyzed using the Agilent Cytogenomics software program v.5.1. (Agilent Technologies Inc., Palo Alto, CA, USA).

Genomic DNA was extracted from peripheral blood samples using the QIAamp Blood Midi Kit (QIAGEN, Hilden, Germany). We performed trio sequencing using a TruSight One sequencing panel consisting of 4813 genes associated with known Mendelian genetic disorders on a MiSeq instrument (Illumina). Sequence data were analyzed using CLC Genomics Workbench version 8.0 (CLC bio, Aarhus, Denmark). Variants detected by MiSeq were validated by conventional Sanger sequencing. Microarray comparative genomic hybridization was performed with the SurePrint G3 Human CGH Micro-array kit 8 × 60 K, Reference DNA Female, and the SureScan Microarray Scanner (Agilent Technologies, Santa Clara, CA, United States). Results were analyzed by CytoGenomics Software version 4.0 (Agilent).