Genomic DNA from triplicate samples was harvested by first washing the plates with cell lysis solution (0.25% Triton X-100, 20 µg/ml RNase H, 10 mM Tris pH 8.0, 1 mM EDTA). Thereafter, nuclei were washed (200 mM NaCl, 20 µg/ml RNase H, 10 mM Tris pH 8.0, 1mM EDTA), prior to lysis with nuclear lysis buffer (0.5% SDS, 200 mM NaCl, 20 µg/ml RNase H, 10 mM Tris pH 8.0, 1mM EDTA). The nuclear lysate was incubated at 37°C for 1 hour. Proteinase K was added to 100 µg/ml and the samples were incubated overnight at 55°C. On the next day the samples were extracted twice using phenol-chloroform-isoamyl alcohol (PCIA, 25:24:1, pH 8.0, from Sigma) and washed twice with chloroform, prior to ethanol precipitation. The pellet was re-suspended in TE (10 mM Tris pH 8.0, 1 mM EDTA).
Label
Cy5 and Cy3
Label protocol
Standard Illumina Protocol
Hybridization protocol
bisulphite converted DNA was amplified, fragmented and hybridised to Illumina Infinium Human Methylation450 Beadchip using standard Illumina protocol
Scan protocol
Arrays were imaged using BeadArray Reader using standard recommended Illumina scanner setting
Data processing
CpG methylation analysis using Illumina 450K Bead Arrays was performed by Genome Quebec. DNA methylation was measured using the Minfi package in-R (Aryee et al., 2014) with the SWAN normalization method. Probes whose total intensity was less than 5000 were excluded. A summary of beta values following the different treatments
