Genome studio methylation module v 1.9.0

DNA samples from control to Aza-treated MCF7 and TMX2-28 cells were sent to the University of Southern California for methylation analysis (HM450 BeadChip; Illumina Cat. No. WG-314-1003). Briefly DNA was quantified using an Alu PCR reaction, bisulfite-treated and quantified by additional PCR reactions prior to running on the array. Then the DNA was enzymatically fragmented at 37 °C for 1 h and precipitated in 100% 2-propanol at 4 °C for 30 min followed by centrifugation at 3000×g at 4 °C for 20 min. After resuspension in hybridization buffer of dried pellets, samples were incubated at 48 °C for 1 h followed by 95 °C for 20 min after which the samples were loaded onto the HM450 BeadChip and incubated at 48 °C for 16–24 h. After hybridization of DNA to the primers on the BeadChip, wash buffers were used to remove non-specific and unhybridized DNA. A single-base extension of the hybridized primers was then conducted using labeled nucleotides and the BeadChip was stained with Cy-3 and Cy-5 fluorescent dyes. The BeadChip was then read using the Illumina iScan Reader. Illumina Genome Studio Methylation Module (v 1.9.0) was then used to analyze the image data to determine the efficiency of the reaction. The ratio of the fluorescent signals of methylated to unmethylated sites (beta values) was used to calculate the methylation of interrogated CpG loci.