Genomic dna uls labeling kit

DNA isolated from fresh/frozen cells was enzymatically labeled using the SureTag Labeling System (Agilent, Santa Clara, CA, USA) and DNA from FFPE tumor tissue was labeled using the Genomic DNA ULS Labeling Kit (Agilent) as described previously^{32 (link)}. Sex-matched human genomic DNA (Promega, Madison, WI, USA) was used as reference. Labeled DNA was hybridized to SurePrint G3 CGH 8X60K microarrays (Agilent). Microarrays were scanned with microarray scanner G2565BA (Agilent) and microarray images were processed by Feature Extraction software version 10.7.3.1 (Agilent). Data were visualized and analyzed using Nexus Copy Number software version 9.0 (BioDiscovery, Inc., El Segundo, CA, USA). Clustering was done using the average linkage hierarchical clustering algorithm. The percentage of genome detected concordantly as gain, loss or neutral between parental tumor and derived cell line was calculated (overlap/overlapping calls).

Molecular serotyping of pneumococci was performed by DNA microarray. An aliquot of the nasopharyngeal swab was inoculated onto Horse Blood Agar supplemented with gentamicin (5 μg/ml), to select for pneumococci, and incubated overnight at 37°C with 5% CO₂. For plates with α-hemolytic growth, the bacterial growth was collected using 1 ml PBS, pelleted by centrifugation and stored at -30°C. DNA was extracted from thawed bacterial pellets using the QIAcube HT with the QIAamp 96 DNA QIAcube HT Kit (Qiagen) with the inclusion of a pre-treatment lysis step whereby 180 μl lysis buffer (20 mM TrisHCl, 2 mM EDTA, 1% Triton X-100, 2 mg/ml RNase A, 20 mg/ml lysozyme) was added to the bacterial pellet and incubated at 37ºC for 60 min. The remaining extraction procedure was as per the manufacturer’s instructions. This DNA was then used for microarray as described previously [12 (link)]. In brief, 200 ng of DNA was labelled with Cy3 or Cy5 using the Genomic DNA ULS Labeling Kit (Agilent Technologies) and incubated at 85°C for 30 min. The labelled pneumococcal DNA was incubated with Senti-SPv1.5 microarray slides (BUGS Bioscience) overnight at 65°C rotating at 20 rpm. Microarray slides were washed, scanned, and analyzed using the Agilent microarray scanner and feature extraction software. Serotype calls were analyzed by Senti-NET software (BUGS Bioscience) using Bayesian-based algorithms.

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.

Indium tin oxide-coated glass slides (50 × 24 × 0.175 mm) with an ITO thickness of 17 ± 2 nm and a sheet resistance of 1200 ± 200 Ω/sq were obtained from Hans Tafelmaier Dünnschicht-Technik GmbH (Rosenheim, Germany). Me-O-PEG-(CH2)₃-Si(OMe)₃ with a MW of 460–590 D was bought from ABCR (#SIM6492.7, Karlsruhe, Germany). Avidin-Cy3 conjugate (#A4500-20) was purchased from USBiological (Swampscott, USA). Genomic DNA ULS Labeling Kit (#5190-0419) was supplied by Agilent Technologies (Vienna, Austria). Avidin (#A9275) and Amicon Ultra centrifugal filter untis (#Z648043-24EA) were obtained from Sigma-Aldrich Handels GmbH (Vienna, Austria). GelRed™ Nucleic Acid Gel Stain (#41003) was supplied by VWR International and Agarose NEEO ultra-quality (#2267.2) was purchased by Carl Roth GmbH (Karlsruhe, Germany). M13mp18 single-stranded DNA was purchased from NEB (Ipswich, USA). Conjugated DNA strand 5′-Biotin-TEG-/CTC GCT TCT GTC TAT CTT GGC-3′ were synthesized by Integrated DNA Technologies (Leuven, Belgium).