Agilent cgh analytics software v3.4

Here, we performed array-based comparative genomic hybridization to demonstrate that there was no chromosome aberration after the CRISPR/Cas9-mediated genome-editing intervention. The genomic DNA of iPSCs with a low passage number (4th–7th passage) was isolated and intermittently sonicated using a Digital Sonifier 450 sonicator probe (Branson Ultrasonics, Danbury, CT, USA). DNA samples were amplified using the GenomePlex WGA kit (Thermo Fisher Scientific, Waltham, MA, USA). A Genomic DNA ULS Labeling Kit (Agilent) was used to label the amplified DNA with either Cy3 or Cy5. As recommended by Agilent, 2.0–2.5 g of amplified DNA were used as the input starting material for each labeling reaction. Scanning and image analysis were conducted according to the Agilent Oligonucleotide Array-based CGH for Genomic DNA Analysis Protocol (version 4.0). Microarrays were scanned using an Agilent G2565BA DNA Microarray Scanner (Agilent). Agilent Feature Extraction software (v9.1.3) was used to extract data from raw microarray image files. Agilent CGH Analytics software (v3.4) was used to visualize, detect, and analyze the aberration patterns from CGH microarray profiles.

Genomic DNA was isolated and intermittently sonicated using a Digital Sonifier 450 sonicator probe (Branson Ultrasonics, Danbury, CT, USA). DNA samples were amplified using the GenomePlex WGA kit (Thermo Fisher Scientific). Genomic DNA ULS Labeling Kit (Agilent) was used to label the amplified DNA with either Cy3 or Cy5. As recommended by Agilent, 2.0–2.5 μg of amplified DNA was used as the input starting material for each labeling reaction. Scanning and image analysis were conducted according to Agilent Oligonucleotide Array-based CGH for Genomic DNA analysis Protocol (version 4.0). Microarrays were scanned using an Agilent G2565BA DNA Microarray Scanner (Agilent). Agilent Feature Extraction software (v9.1.3) was used to extract data from raw microarray image files. Agilent CGH Analytics software (v3.4) was used to visualize, detect and analyze the aberration patterns from CGH microarray profiles.

Array CGH was performed using a human genome-wide 185K oligonucleotide array (Agilent Technologies). Genomic DNA from the inversion patient (II-4, family #1) and from a sex-matched reference were double-digested separately using the restriction endonucleases AluI and RsaI (Promega) and purified using Microcon centrifugal filter devices (Merck Millipore). A total of 1.5 μg of the digested products was differentially labelled by the random priming method using the fluorophores Cy3-dUTP and Cy5-dUTP (Perkin Elmer) and co-hybridised to the array for 48 h at 65°C in a rotating oven. The hybridised arrays were washed and scanned using an Agilent Microarray Scanner. The image data were extracted using Agilent Feature Extraction software V.8.5, and the data analysed using Agilent CGH Analytics software V.3.4 (z-score method setting).