Epigenetic Clock Protocol from Cord Blood

Samples were processed as previously described [30 (link)]. Briefly, DNA was extracted from umbilical cord blood samples using QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Mettmann, Germany), according to the manufacturer’s instructions. DNA was bisulfite-converted using the EZ DNA Methylation kit (Zymo Research, Irvine, California, USA), according to manufacturer’s instructions and hybridized in the Illumina Infinium HumanMethylation450 BeadChip array (Illumina Inc., San Diego, California, USA). Raw data was extracted by iScan SQ scanner (Illumina) with GenomeStudio software (v.2011.1), using the methylation module v.1.9.0 (Illumina), into IDAT files used for further analyses. Raw data files were normalized using Quantile and corrected for cell composition using a cord blood dataset as implemented in minfi. Multidimensionality graphs (MDS) were also used to obtain the biological variability of the data. The X and Y chromosome probes were excluded to avoid sexual identity bias. Non-specific probes were excluded due to the high probability of co-hybridization. Single Value Decomposition method (SVD) identified technical bias (slide), which was corrected using ComBat. Probes from HM450K were annotated according to their nearest genes using FDb.InfiniumMethylation.hg19 package. Our main interest here with DNAm data was obtaining the epigenetic clock (EC).