Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood using the Ficoll density gradient separation. DNA and RNA were isolated simultaneously from the PBMCs using the AllPrep DNA/RNA kit (Qiagen, Germantown, MD), DNA and RNA samples were quantified and purity assessed using a NanoDrop spectrophotometer (Thermo Scientific, Wilmington, DE). RNA integrity was determined using the Bioanalyzer (Agilent, Santa Clara, CA).
Label
Cy3
Label protocol
0.85-1.00 µg DNA were bisulfite converted using the Zymo EZ DNA Methylation kit (Zymo Research, Orange, CA). Each conversion assay included a commercially available positive and negative control (Universal Methylation DNA Standard, Zymo Research, Orange, CA). Bisulfite converted samples formed the input for the Illumina Infinium Methylation assay
Hybridization protocol
Illumina Infinium protocol
Scan protocol
Illumina iScan
Data processing
normalized using the SWAN method contained in the R package minfi; the normalized M-values were used in all downstream analyses Unmethylated and methylated signal intensities are reported in matrix-raw-values.txt which is linked as a supplementary file on the Series record.
