Infinium humanmethylation450k beadchip array

DNA was extracted and processed at the Epigenetics Group, IARC. Specifically, DNA was bisulfite-converted using the Zymo EZ DNA methylation^TM kit (Zymo, Irvine, CA, United States). DNA was then hybridized to Illumina Infinium Human Methylation 450K BeadChip arrays (66) and scanned using the Illumina HiScanSQ system. After background subtraction using Illumina GenomeStudio raw intensity data were submitted to pre-processing, including normalization, using in-house software within the R statistical computing environment. Furthermore, quality control of samples was carried out and failed samples were excluded on the basis of Illumina’s detection p-value greater than 0.01 and bead count lower than 3. For probes using the Infinium II design additional background subtraction and dye bias correction were performed. Methylation levels at each CpG locus were expressed by Beta-values, as defined by the ratio of signal intensity originating from methylated CpGs over the sum of methylated and unmethylated CpGs. Finally, data were trimmed for outliers with values larger than 3 interquartile ranges below the first quartile or above the fourth quartile. CpG sites were annotated using the Bioconductor package IlluminaHumanMethylation450kanno.ilmn12.hg19 in R for the annotation of Illumina’s 450K methylation arrays.

Cord blood DNA was extracted and processed at the Epigenomics and Mechanisms Branch (formerly Epigenetics Group), International Agency for Research on Cancer (IARC). In detail, after thawing and extraction with the QIAamp DNA mini Kit (Qiagen Ltd, Manchester, UK), DNA was first bisulfite-converted using the Zymo EZ DNA methylation^™ kit (Zymo, Irvine, CA, USA), consequently hybridized to Illumina Infinium Human Methylation 450K BeadChip arrays [45 (link)] and scanned using the Illumina HiScanSQ system. After background subtraction with Illumina GenomeStudio, the raw intensity data were preprocessed, including the calculation of the methylation level at each CpG as the beta-value, the normalization using the funnorm normalization of the minfi package [46 (link)], and quality control employing in-house software within the R statistical computing environment. Samples underwent further quality control employing Illumina’s detection p-value > 0.05 and bead count lower than 3, excluding failed samples. Additionally, background subtraction and dye bias correction were performed on Infinium II probes. Finally, data were also trimmed for outliers containing values more than three interquartile ranges below the first quartile or above the third quartile so that 485,512 probes remained for analysis.

Samples for leptin were batched and measured in duplicate with a radioimmunoassay kit (Millipore Corp, Billerica, MA, USA). The inter- and intra-assay coefficients of variation for leptin were 3.7–5.9% and 3.0–4.0%, respectively.
DNA was purified from neonatal cord blood using an Autopure LS Automated DNA Purification System with Autopure reagents (Autogen, Inc., Holliston, MA). The purified DNA samples were stored under -20°C. DNA quality and quantity were assessed by Nanodrop (ThermoFisher Scientific, MA, USA). Bisulfite conversion was performed on 500 ng of DNA using the EZ DNA Methylation Kit (Zymo Research, CA, USA). Methylation levels were measured using the Infinium HumanMethylation 450K Beadchip array (Illumina, Inc. CA, USA), which targets ~486,000 CpG sites, in 114 samples that passed DNA quality testing. Samples were randomly plated on each chip with regard to neonatal sex. BeadChips were scanned with an Illumina iScan and analyzed using Illumina GenomeStudio software. All experiments were conducted following manufacturer protocols in the Genomics Core Facility at the Center for Genetic Medicine at Northwestern University.