Human methylation 450k beadchip

Fragmented DNA was dispensed onto the multichannel HumanMethylation450K BeadChips and hybridization performed in an Illumina Hybridization oven for 20 h. BeadChips were washed, primer extended, and stained per manufacturer protocols. BeadChips were coated and then imaged on an Illumina iScan Reader and images were processed with GenomeStudio software methylation module (v. 1.8 or later; Illumina).

A 1-cm³ sample of the placenta near the fetal surface was collected, and genomic DNA extracted. DNA was assessed by agarose gel electrophoresis with ethidium bromide staining and by PicoGreen assays (Life Technologies, Carlsbad, Calif.). DNA, 500 ng, was bisulfite converted and analyzed according to the manufacturer’s instructions for Illumina Human Methylation 450K BeadChips, with all assays performed at the Roswell Park Cancer Institute Genomics Shared Resource. Data were processed using Genome Studio software, which calculates the fractional methylation (AVG_Beta) at each CpG, after background correction and normalization to internal controls. All probes querying CpGs that overlapped common single-nucleotide polymorphisms (SNPs) (SNPs with minor allele frequency ≥1% in dbSNP build 138) were removed. Bisulfite sequencing was performed by converting the DNA samples using the EpiTect Bisulfite kit (Qiagen, Hilden, Germany), amplifying the DNA by polymerase chain reaction (PCR), cloning using the TOPO TA Cloning kit (Life Technologies, Carlsbad, Calif.), with three independent PCRs pooled for each sample prior to bacterial transformation, and sequencing of multiple clones.

DNAm from whole blood was quantified at 485,512 CpG sites using the Illumina Human Methylation 450k BeadChips at the Edinburgh Clinical Research Facility. Full details of the quality control steps have been described previously (Shah et al., 2014 (link); Zhang et al., 2018 (link)). Briefly, raw intensity data were background-corrected and normalised using internal controls. Samples with inadequate bisulphite conversion, hybridisation, staining signal or nucleotide extension were removed upon manual inspection. Further, probes with a low detection rate (p>0.01 in >5% of samples), samples with a low call rate (<450,000 probes detected at p<0.01), samples exhibiting a poor match between genotype and SNP control probes, and samples with a mismatch between methylation-predicted and recorded sex were additionally excluded. This left a total of 450,276 autosomal probes. In analyses comparing blood and brain DNAm signatures, the last blood measurement before death was used and models were adjusted for the interval between the blood draw and death (see Supplementary Table 1; mean interval: 2.5 years, SD: 1.5).