Genomic DNA was extracted from peripheral blood leukocytes using standard procedures. DNA quality and quantity were assessed by NanoDrop (Thermo Fisher Scientific), Qubit (Thermo Fisher Scientific), and electrophoresis on 1% agarose gel. The bisulfite-converted DNA (EZ DNA Methylation kit, Zymo Research) was hybridized in the Human Methylation 450 BeadChip microarray (HM450K, Illumina), following the Illumina Infinium HD methylation protocol. We used RnBeads tools to evaluate the quality of our data, and all samples provided high-quality data [53 (link)]. Briefly, experimental quality control was performed using the microarray positive and negative control probes for staining, hybridization, extension, target removal, bisulfite conversion, specificity, and non-polymorphic sites.
Data were extracted by the iScan SQ scanner (Illumina) using GenomeStudio software (v.2011.1), with the methylation module v.1.9.0, into IDAT files.
Probes were annotated using GRCh37/hg19 coordinates from UCSC regarding genomic positions and features (FDb.InfiniumMethylation.hg19 package), with additional annotations to identify probes that exhibit multiple alignments in the genome for posterior exclusion.
Methylation levels of the CpG sites were calculated as beta values, which range continuously from 0 (unmethylated) to 1 (fully methylated) (http://www.illumina.com ).
Data were extracted by the iScan SQ scanner (Illumina) using GenomeStudio software (v.2011.1), with the methylation module v.1.9.0, into IDAT files.
Probes were annotated using GRCh37/hg19 coordinates from UCSC regarding genomic positions and features (FDb.InfiniumMethylation.hg19 package), with additional annotations to identify probes that exhibit multiple alignments in the genome for posterior exclusion.
Methylation levels of the CpG sites were calculated as beta values, which range continuously from 0 (unmethylated) to 1 (fully methylated) (
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