For the set of genomic regions that were also used in transient enhancer-reporter assays, PCR primers were designed using the MethPrimer web tool57 and purchased from Sigma-Aldrich (Munich, Germany) (for sequences see Supplementary Table 7). Sodium bisulfite conversion was performed using the EZ DNA methylation kit (Zymo Research, California, USA) using 200-1000 ng of genomic DNA from CD4+CD25− T cells, CD8+ T cells, CD14+ monocytes, CD19+ B cells, and CD56+ NK cells (two donors each) and an alternative conversion protocol. Amplification of target regions was followed by SAP treatment, reverse transcription and subsequent RNA base-specific cleavage (MassCLEAVE, San Diego, CA) as previously described58. Cleavage products were loaded onto silicon chips (SpectroCHIP, CA) and analyzed by MALDI-TOF mass spectrometry (MassARRAY Compact MALDI-TOF, U.S.Sequenom, San Diego, CA). Methylation was quantified from mass spectra using the Epityper software (Sequenom, U.S), and averaging methylation levels of CpG dinucleotides located in the central DNase hypersensitive (nucleosome-free) region that is flanked by CAGE clusters. The methylation data for individual CpGs are provided in Supplementary Table 8.
