Agilent genomic dna labeling kit plus

Genomic DNA was quantified by spectrophotometry (NanoDrop ND1000, NanoDrop Technologies, Wilmington, Delaware USA). Integrity of DNA was assessed by 0.8% agarose gel electrophoresis. Non-amplification labeling of DNA (direct method) was obtained following the ‘Agilent Oligonucleotide Array-Based CGH for Genomic DNA Analysis’ protocol Version 4.0 (Agilent Technologies, Palo Alto, California USA. p/n G4410-90010). 500 ng of experimental and pool female reference genomic DNA samples were fragmented in a restriction digestion step. Digestion was confirmed and evaluated by DNA 7500 Bioanalyzer assay. Cyanine 3-dUTP and cyanine 5-dUTP were used for fluorescent labeling of test and reference digested gDNAs respectively, using the ‘Agilent Genomic DNA Labeling Kit PLUS’ (Agilent p/n 5188-5309) according to the manufacturer's instructions. Labeled DNA was hybridized with Human Genome CGH Microarray 44K (Agilent p/n G4426B-014950) containing 43,000+ coding and noncoding human sequences. Arrays were scanned in an Agilent Microarray Scanner (Agilent G2565BA) according to the manufacturer's protocol and data extracted using Agilent Feature Extraction Software 9.5.3.1 following the Agilent protocol CGH-v4_95_Feb07 (‘Lowess Only’ normalization correction dye bias method instead of ‘Linear Only’) and the QC Metric Set CGH_QCMT_Feb08.

All gcCGH tests were performed, analyzed and validated based on the protocols described previously [26] (link), [27] (link). In brief, all DNA samples were assessed for genomic DNA concentration and purity. Agarose gel electrophoresis was used to assess the quality of the genomic DNA samples. The test DNA (3 µg) and reference DNA (3 µg, pooled normal human male or female DNAs were used as reference DNA; purchased from Promega, Madison, WI) were digested with AluI and RsaI (Promega), and then labeled using the Agilent Genomic DNA Labeling Kit PLUS (Agilent Technologies, Santa Clara, CA). The individually labeled test and reference samples were then purified using Microcon YM-30 filters (Millipore, Billerica, MA). Following purification, the appropriately labeled test DNA and reference DNA were mixed together and hybridized to the custom gcCGH array. The hybridization was followed by four washing steps, and slides were scanned on an Agilent Microarray Scanner G2565BA with 5-µm resolution. Captured images were assessed with Feature Extraction Software, version 9.5 (Agilent), and the data were then imported into Agilent CGH Analytics 3.2.5 software for statistical analysis.

Genomic DNA was extracted from HCC21 cells and the original tumor. The Agilent human 44 K CGH microarray (Agilent Technologies, Santa Clara, CA) with an average probe spatial resolution of ∼ 35 kb was utilized. Genomic DNA at 1 μg was labelled by the Agilent Genomic DNA Labeling Kit PLUS (Agilent Technologies). The hybridized array was scanned by an Agilent Microarray Scanner and data were analysed by Agilent Feature Extraction 9.1 followed by computation and normalization using CGH Analytics v3.4. Putative chromosome copy number loss was defined by intervals of two or more adjacent probes with log₂ ratios suggestive of a deletion when compared with the log₂ ratios of adjoining probes. The Quality-Weighted Interval Score algorithm (ADM2) with a value of 6.0 was used to compute and assist the identification of aberrations for a given sample.

Formaldehyde crosslinking and chromatin immunoprecipitation (ChIP) assays were performed with primary antibodies against histone H3K4me3 (EMD Millipore, Billerica, MA, USA) using a Magna ChIP™ A chromatin immunoprecipitation kit (EMD Millipore). Sheared by sonication and purified, immunoprecipitated DNA and input DNA were amplified by a GenomePlex® Complete Whole Genome Amplification kit (Sigma-Aldrich, St. Louis, MO, USA). Amplified immunoprecipitated DNA and input DNA samples were labeled with cyanine 3-dUTP and cyanine 5-dUTP, respectively, using an Agilent Genomic DNA Labeling Kit Plus (Agilent Technologies, Santa Clara, CA, USA). The labeled DNA samples were purified using Amicon Ultra-0.5 30 kDa columns (Millipore, Billerica, MA, USA) and eluted with 22 μL of 10 mM Tris, 1 mM EDTA buffer, pH 8.0. The labeled immunoprecipitated DNA and input DNA were co-hybridized to Agilent Mouse 2 × 105 K CpG Island Microarrays according to the manufacturer’s protocol. Microarrays were scanned on an Agilent DNA microarray scanner and the resulting images were analyzed with Agilent Feature Extraction Software (Version 10.7.3). The data files were loaded into the ArrayTrack database [37 (link)] for Lowess normalization, calculation of the ratio of immunoprecipitated/input DNA, and further statistical analyses.