DNA from FF tumours was extracted with the DNeasy Blood & Tissue Kit (69504, Qiagen, Hilden, Germany) according to the manufacturer’s instructions with the addition of lysing the samples with the QIAGEN TissueLyser. FFPE samples (1-3 years old) were sectioned and DNA was isolated using QIAamp DNA FFPE Tissue Kit (56404, Qiagen) according to manufacturer’s recommendations except de-waxing steps with Xylene was repeated twice and the tissue was digested overnight. DNA concentration was determined with the Qubit® Fluorometer (Life technologies). The quality of the DNA extracted from FFPE samples was assessed in triplicates with the real-time PCR-based Illumina FFPE QC kit (WG-321-1001, Illumina, Inc., San Diego, CA) according to the provided protocol.
Label
Cy5 and Cy3
Label protocol
500 ng FF DNA, 1 µg REPLI-g restored FFPE DNA and 1 µg unrestored FFPE DNA (for restoration with the Infinium kit after bisulfite conversion) was used for bisulfite conversion with the EZ DNA methylation kit (D5001, Zymo Research, Orange, CA) according to the manufacturer’s instructions using the alternative incubation conditions recommended for the Illumina Infinium methylation arrays. Successful conversion was verified by control PCR reactions with a primer set specific for bisulfite converted DNA and a primer set for unconverted DNA.
Hybridization protocol
Bisulphite converted DNA was amplified, fragmented and hybridised to Illumina Infinium MethylationEPIC Beadchips using standard Illumina protocol
Scan protocol
standard Illumina protocol
Data processing
Raw methylation data was normalized using ssNoob-normalization with the R minfi package. CpG-sites with detection p-value >0.01 were regarded as failed and were assigned as missing.