Genomic DNA was extracted from all LC-C2 tissue samples and lung cancer cell lines using a QIAamp DNA mini kit (Qiagen, Valencia, CA, USA). Bisulfite conversion using an EZ DNA Methylation-Gold kit (Zymo Research, Irvine, CA, USA) was carried out on 500 ng aliquots of DNA. Subsequently, DNA methylation status at 27,578 CpG loci was examined at single-CpG resolution using the Infinium HumanMethylation27 Bead array (Illumina, San Diego, CA, USA). An Evo robot (Tecan, Männedorf, Switzerland) was used for automated sample processing. After whole genome amplification and hybridization, the specifically hybridized DNA was fluorescence-labeled by a single-base extension reaction and detected using a BeadScan reader (Illumina) in accordance with the manufacturer's protocols. The data were then assembled using GenomeStudio methylation software (Illumina). At each CpG site, the ratio of the fluorescence signal was measured using a methylated probe relative to the sum of the methylated and unmethylated probes, such as the β-value, which ranges from 0 to 1 and reflects the methylation level of an individual CpG site. The reliability of DNA methylation levels (β-values) determined by the Infinium assay was verified in our previous studies (12 (link)–14 (link)).
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