Epityper t complete reagent set

Genomic DNA was isolated from the purified cell pellets using the QIAmp DNA Mini Kit (Qiagen) according to the manufacturer’s protocol. DNA from whole blood was extracted by the salting-out method using 10 M ammonium acetate. The DNA was precipitated in isopropanol, washed in 70% ethanol, and finally resuspended in 1X TE buffer. The purity and concentrations of the DNA samples were measured by NanoDrop ND-1000 spectrophotometry. 500 ng of genomic DNA was treated with sodium bisulfite using the EZ DNA Methylation Kit (Zymo Research Corporation) according to the manufacturer’s instructions. DNA methylation analysis was performed using the Infinium Human Methylation 450 K BeadChip (Illumina). Quantitative analysis of DNA methylation was verified using Sequenom’s EpiTYPER T Complete Reagent Set and MassARRAY Analyzer 4 (Sequenom). DNA was amplified using bisulfite-converted DNA, Hot Start DNA Polymerase (Solis BioDyne) and specific primers, according to the EpiTYPER protocol. Primers were designed with Sequenom’s EpiDesigner program and are listed in Supplementary Table S4.

Quantitative DNA methylation analyses of the enhancers in the TMEM241 and GFPT2 genes was performed using Sequenom's MassARRAY platform. Bisulfite‐treated libraries were PCR‐amplified. The PCR product was in vitro transcribed and cleaved by RNase A using the EpiTyper T Complete Reagent Set (Sequenom, Hamburg, Germany) and subjected to MALDI‐TOF mass spectrometry analysis to determine methylation patterns as previously described (Ehrich et al, 2008). DNA methylation standards (0, 20, 40, 60, 80, and 100% methylated genomic DNA) were used to control for potential PCR bias.
For qPCR, total RNA was prepared from fresh cord blood by using peqGold RNA Pure (peqlab, Erlangen, Germany) and from blood collected in PAXgene Blood RNA Tube of year four by PAXgene Blood RNA Kit (Qiagen, Hilden, Germany), according to manufacturer's instructions. The cDNA synthesis was carried out with 5 μg of RNA by using ImProm‐II^™ Reverse Transcription System (Promega, Mannheim, Germany). Gene expression was measured using the 96.96 Dynamic Array or FLEXsix Integrated Fluidic Circuits (IFCs) (Fluidigm, San Francisco, CA, USA). Intron‐spanning primers were designed (see Appendix Supplementary Methods). Gene expression values were determined by using the 2^−∆∆CT method (Livak & Schmittgen, 2001) with GAPD and GUSB, as reference genes and normalized to the lowest measured value.