Genomic DNA for the Infinium methylation assay was extracted using phenol-chloroform and ethanol precipitation. DNA concentration was determined using a Nanodrop ND-1000 spectrophotometer (Thermo Scientific, Wilmington, DE), and DNA extraction was repeated using a new tissue aliquot for samples with DNA concentration less than 50ng/ul, or with a 260/280 ratio less than 1.7.
Label
C-Bio and A-DNP
Label protocol
1ug of genomic DNA underwent bisulfite conversion using the WZ-96 DNA Methylation Kit according to the manufacturer’s protocol (Zymo Research Corp, Orange, CA). Incubation conditions used during conversion were as follows: (95°C for 30 seconds, 50°C for 1 hour) for 16 cycles, hold overnight at 4°C. Unmethylated cytosines were chemically deaminated to uracil in the presence of bisulfite, while methylated cytosines were refractory to the effects of bisulfite and remained as cytosines. Methylation was then detected as a C/T nucleotide polymorphism at each CpG site. After bisulfite conversion, each sample was whole-genome amplified, fragmented, and hybridized to the BeadChip. DNA molecules anneal to locus-specific DNA oligomers.
Hybridization protocol
Two bead types correspond to each CpG locus, one to the methylated (C) state, and the other to the unmethylated (T) state. After extension, the BeadChip was fluorescently stained.
Scan protocol
Illumina Beadstation BeadArray laser confocal scanner to measure the intensities of unmethylated and methylated bead types for each locus.
Description
Human Brain Tissue: PONS
Data processing
Beadstudio Methylation Module v3.2.0, no normalization (analysis performed on the non-normalized data).
