Genomic DNA were extracted and purified from lung cancer and paired non-cancerous tissues using the QIAamp DNA Mini kit(Qiagen, Hilden, Germany) according to the manufacturer’s instructions.
Label
Cy5
Label protocol
A total of 6 µg of the pooled gDNA from each tissue type was digested by MseI, purified by using QIAquick PCR purification kit (Qiagen), and analyzed by agarose gel electrophoresis. MseI-digested gDNA (1.25 µg) was subjected to immunoprecipitation with a mouse monoclonal anti-5-methylcytosine antibody (Diagenode, Liège, Belgium) and then was purified through Qiagen MinElute columns and amplified by using a GenomePlex Complete Whole Genome Amplification (WGA2) kit from Sigma-Aldrich (St Louis, MO, USA). The amplified DNA samples were purified with a QIAquick PCR purification kit. The NimbleGen Dual-Color DNA Labeling kit (Roche NimbleGen Systems, Inc., Madison, WI, USA) was used for labeling according to the manufacturer’s guidelines. Briefly, 1 µg DNA of each sample was incubated for 10 min at 98°C with 1 OD of Cy5-9mer primer (immunoprecipitation sample) or Cy3-9mer primer (input sample). Then, 100 pmol of deoxynucleoside triphosphates and 100 U of the Klenow fragment (New England Biolabs, Ipswich, MA, USA) were added, and the mix was incubated at 37°C for 3 h.
Genomic DNA were extracted and purified from lung cancer and paired non-cancerous tissues using the QIAamp DNA Mini kit(Qiagen, Hilden, Germany) according to the manufacturer’s instructions.
Label
Cy3
Label protocol
A total of 6 µg of the pooled gDNA from each tissue type was digested by MseI, purified by using QIAquick PCR purification kit (Qiagen), and analyzed by agarose gel electrophoresis. MseI-digested gDNA (1.25 µg) was subjected to immunoprecipitation with a mouse monoclonal anti-5-methylcytosine antibody (Diagenode, Liège, Belgium) and then was purified through Qiagen MinElute columns and amplified by using a GenomePlex Complete Whole Genome Amplification (WGA2) kit from Sigma-Aldrich (St Louis, MO, USA). The amplified DNA samples were purified with a QIAquick PCR purification kit. The NimbleGen Dual-Color DNA Labeling kit (Roche NimbleGen Systems, Inc., Madison, WI, USA) was used for labeling according to the manufacturer’s guidelines. Briefly, 1 µg DNA of each sample was incubated for 10 min at 98°C with 1 OD of Cy5-9mer primer (immunoprecipitation sample) or Cy3-9mer primer (input sample). Then, 100 pmol of deoxynucleoside triphosphates and 100 U of the Klenow fragment (New England Biolabs, Ipswich, MA, USA) were added, and the mix was incubated at 37°C for 3 h.
Hybridization protocol
The labeled DNA was purified by isopropanol/ethanol precipitation and hybridized to NimbleGen Human Meth 3x720K CpG RfSq Prom according to the manufacturer’s instructions.
Scan protocol
Signal intensity data were extracted from the scanned images of each array by NimbleScan MS 200 Microarray Scanner and the MS 200 Data Collection Software. Median-centering, quantile normalization, and linear smoothing were performed by the Bioconductor software packages Ringo, limma, and MEDME, respectively.
Data processing
Normalized log2-ratio data were created for each sample, and a sliding-window peak-finding algorithm provided by NimbleScan v2.5 was applied to determine the enriched peaks with specified parameters (sliding window width: 750 bp; miniprobes per peak: 2; p-value minimum cutoff: 2; maximum spacing between nearby probes within peak: 500 bp). A one-sided Kolmogorov–Smirnov test was applied to determine whether the probes were drawn from a significantly more positive distribution of intensity log2-ratios than those in the rest of the array. Each probe received a −log10 p-value score from the windowed Kolmogorov–Smirnov test around that probe. If several adjacent probes rose significantly above a p-value minimum cutoff (−log10) of 2, the region was considered to be an enrichment peak. When comparing two groups' differentially enriched regions, we averaged the log2-ratio values for each group (Experiment and Control) and calculated M′ value for each probe, where M′=Average (log2 MeDIPE/InputE) – Average (log2 MeDIPC/InputC). Then the NimbleScan sliding-window peak-finding algorithm was rerun on these data to find the differential enrichment peaks.
