Genomic dna labeling kit plus

ChIP was done according to the Upstate protocol (Smith et al. 2010 (link)). Input DNA and IP DNA (10 ng) were amplified and labeled according to the Agilent Genomic DNA Labeling Kit PLUS (G4481-90010). Custom Agilent 2×105K Hox tiling arrays were hybridized with a mixture of 4 µg Cy3 labeled DNA and 4 µg Cy5 labeled DNA probes. Hybridizations were performed for 24 h at 65°C under standard conditions. Microarray images were acquired with an Agilent High-Resolution DNA Microarray Scanner (G2505C). For image analysis, Agilent Feature Extraction software (Version 10.5.1.1) was used. Agilent tiling arrays were hybridized in a two-color configuration. Data were analyzed using the limma (3.4.3) package and normalized within arrays using loess normalization.

To investigate DNA copy number changes on bulk tumors, we used the Mouse 244 k Custom Oligo platform (GPL15359 Agilent UNC Perou Lab 1 × 244 k Custom Tiling CGH Array)^{69 (link)}. Labeling and hybridization were performed according to the manufacturer’s instructions using the Agilent Genomic DNA Labeling Kit PLUS (Catalog Number 5188–5309). One microgram of DNA from liver or spleen of FVB strain mouse was used as normal reference DNA, which was compared versus 1 μg of DNA from C3Tag DCIS and tumor samples. Microarrays were scanned on an Agilent DNA Microarray scanner (G2565CA) and the data uploaded to the University of North Carolina Microarray Database (www.genome.unc.edu). To determine regions of Copy Number Aberration (CNA), we utilized the R package SWITCHdna^{18 (link),70 (link)}.