Feature extraction v9

Methylated regions of the genome from liver genomic DNA were isolated and labeled as described in the Agilent Microarray Analysis of Methylated DNA Immunoprecipitation protocol (http://www.agilent.com/cs/library/usermanuals/public/G4170-90012_Methylation_2.3.pdf). The labeled DNAs were then hybridized onto the Agilent mouse CpG Island 2×105k microarray (Agilent G4811A). Data was obtained using an Agilent scanner at 5 μm resolution, followed by processing using the Agilent Feature Extraction (v9.5) software.
Examination of the distribution of the raw probe signals and analysis of background revealed that one of the samples was a clear outlier, so this sample and its sibling control were dropped from the analysis (mice pair #2). In addition, probes were removed from the analysis if called absent in less then four of the five remaining pairs or flagged as poor quality in any of the arrays. For the remaining samples, data was log₂ transformed, and the difference between the Cy3 and Cy5 samples was determined. Differential methylation was then assessed using the limma package in Bioconductor. A list of differentially methylated promoter probes between −Zn and +Zn mice was selected on the basis of the probe being located in the promoter and a P<0.01. This list was then used for Gene Ontology Analysis using Web-Gestalt (Wang et al 2013 (link)).

RNA was extracted from indicated tumor cells under CPT (50 μM) treatment and analyzed with Agilent Whole Mouse Genome 4×44k arrays. RNA samples were labeled with Cy5 using the Agilent Low RNA Input Linear Amplification Kit and hybridized along with the Cy3-labeled Mouse Universal Reference RNA (Stratagene). Arrays were scanned with an Agilent G2565BA scanner and analyzed with the Agilent Feature Extraction v9.5 software. The Cy5/Cy3 ratios were calculated using the feature medium signal and normalized by the array median. Genes with > 2 average fold changes and Student’s t-test p values < 0.05 were included as SND1-regualated genes.