Hiscan 2000

Total Genomic DNA was isolated using NucleoSpin® Tissue kit (Macherey-Nagel, cat no.: 740952.250) following the manufacturer’s instructions. Extracted genomic DNA was treated with sodium bisulfite using EZ-96 DNA methylation Gold kit (Zymo Research, cat no.: D5007). Assessment of levels of DNA methylation of known CpG regions and promoters across the genome was done with Illumina HumanMethylation 450K BeadChip and Illumina HiScan 2000. In brief, following bisulfite conversion, approximately 200 ng of the bisulfite-converted DNA per sample was used for methylation analysis. The initial quality control and identification of signal intensities for each probe were performed with Illumina GenomeStudio Software.

Microarray-based DNA methylation profiling was performed using the Illumina Infinium MethylationEPIC (EPIC) BeadChip (Illumina, Inc., San Diego, CA, USA) on 7 paired blood samples. Genomic DNA from peripheral blood samples was extracted using a DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany). Bisulfite conversion of isolated genomic DNA (500 μg) was performed using the EZ DNA methylation Gold Kit (Zymo Research, Irvine, USA). Bisulfite-converted DNA was then whole-genome amplified, enzymatically fragmented, and hybridized to the array as per the EPIC BeadChip protocol^{18 (link)}. Subsequent scanning of chips was performed using an Illumina HiScan2000. The raw intensity of the data was determined using GenomeStudio methylation module version 1.9.0 (Illumina, Inc.).

The 485,577 methylation probes from the Human Methylation450 array were filtered. The DNA methylation was measured and with detection p values above the significance threshold 0.05.
Microarray chips were scanned by a HiScan 2000 (Illumina). To minimize any effects of sample processing, validation cohort arrays were run in the same batch and with the same operators as a subset of the samples from the discovery cohort. This DNA methylation data for the discovery and validation cohorts is available from the Gene Expression Omnibus (GEO) database under the accession numbers GSE100197 and GSE98224, respectively.
Raw data (IDAT Files) were read into R statistical software, version 3.2.4, where functional normalization, background subtraction, and color correction were performed as Blair et al. previously used subset within-array normalization (SWAN). Functional normalization performs all the benefits of SWAN normalization and, in addition, utilizes the 900 control probes on the array to mediate changes in DNA methylation that are due to technical effects [17 (link)].