DNA was extracted from fresh-frozen blocks with an estimated purity of ≥95% using TRIzol (Thermo Fisher Scientific) according to the manufacturer’s protocol.
Label
Cy3, Cy5
Label protocol
Standard Illumina protocol.
Hybridization protocol
We quantified DNA with Picogreen and used 500ng for DNA bisulfite treatment. DNA was amplified for 20-24 hours at 37°C, then hybridized for 16-20 hours at 48ºC.
Scan protocol
Arrays were scanned using Illumina iScan with the Methylation NXT setting. We analyzed methylation data with Genomestudio v2011.1 and Methylation module 1.9.0
Description
20-01-050 DNA methylation profiling (EPIC 850K)
Data processing
Raw data was imported, processed, and normalized using the ChAMP pipeline (ChAMP package, R 4.0.3) using default parameters. Probes were filtered out if they failed to hybridize (detection p-value > 0.01), had <3 beads in 5% of samples, were not at CpG sites, were defined as multi-hit probes, were located on sex chromosomes, or were associated with single nucleotide polymorphisms (ChAMP package) Beta values were calculated and normalized using the beta mixture quantile normalization (BMIQ, ChAMP package) We performed SVD analysis to look for batch effects but found none that warranted correction (ChAMP package)
