Infinium humanmethylation27 beadchip

A hematoxylin & eosin-stained frozen section was prepared for assessment of the percentage of tumor cells before RNA extraction. Only samples with > 80% tumor cells were selected. Genomic DNA was isolated from frozen tumor tissues using the QIAamp DNA Mini Kit (Cat No. 51304, QIAGEN) according to the manufacturer’s protocol. DNA concentration and quality were measured using the NanoDrop ND-1000 spectrophotometer. We used the Illumina Infinium HumanMethylation27 Bead-Chip. The Bead-Chip contains 27,578 highly informative CpG sites covering more than 14,000 human RefSeq genes. This array allows researchers to interrogate all of these sites per sample at a single nucleotide resolution. Bisulfite modification of DNA, chip processing, and data analysis were performed following the manufacturer’s manual at the Wellcome Trust Centre for Human Genetics Genomics Lab, Oxford, UK. The array results were analyzed using the BeadStudio software (Illumina).

DNA methylation profiling was performed with the Infinium HumanMethylation27 beadchip (Illumina Inc.). DNA from GBM samples and control brains were bisulfite-modified, using the EZ DNA methylation kit (Zymo Research) and hybridized according to the manufacturer's instructions. For each interrogated CpG site, methylation status is calculated by dividing the signal from the methylated probe (M) by the sum of signals for both methylated and unmethylated (U) probes (Genome Studio 2008.1, Illumina Inc.): β-value = Max(M, 0)/ [Max(M, 0) + Max(U, 0) + 100]. This β-value provides a continuous and quantitative measurement of DNA methylation, ranging from 0 (completely unmethylated) to 1 (completely methylated). Missing values were imputed by nearest neighbors averaging (impute R package). All samples were G-CIMP negative [9 (link)]. All samples were MGMT unmethylated [52 (link)] except FT06_TZ and FT06_I. Methylation arrays have been deposited in the Gene Expression Omnibus repository under the accession number GSEXXXXX.

The Illumina Infinium HumanMethylation27 BeadChip (27,578 CpG dinucleotides for 14,495 genes) was adapted for DNA methylation detection according to manufacturer’s manual. DNA methylation levels were reported as β-values by calculating the ratio of intensities between locus-specific methylated and unmethylated bead-bound probes. The β-value is a continuous variable, ranging from 0 (unmethylated) to 1 (fully methylated). The methylation array data can be viewed online under GEO accession number GSE83845.

A total of 6 cSCC lines characterised within this manuscript were used for the detection of differentially methylated genes. Three early-passage primary normal human keratinocytes derived from 3 different participants served as a normal control for comparison. DNA was extracted as described above and bisulfite modification was carried out as the following: 500–1000 ng of DNA yield from each sample was modified with bisulfite using the EZ DNA Methylation Kit (Zymo, Research, CA, USA) according to the manufacturer’s instructions. Methylation detection was performed using the Illumina Infinium HumanMethylation27 BeadChip. This platform detects the methylation status of 27,578 CpG sites spread across 14,495 genes by sequencing-based genotyping of bisulfite-converted DNA. Eventual methylation scores (denoted “beta-value”) were generated for each site with BeadStudio software (Illumina, Inc., San Diego, CA, USA) and raw background-corrected values were used for further analysis. The methylation assay was performed according to the manufacturer’s instructions. Briefly, bisulfite-converted DNA was amplified, fragmented and hybridised to the chip arrays, followed by imaging with the Illumina BeadArray reader. Image processing and intensity data extraction were performed according to Illumina’s instructions. All analyses were carried out using Bioconductor and R.