Bisulfite Conversion and Pyrosequencing Protocol

Bisulfite converted DNA was initially subjected to PCR amplification. Primers were purchased at Metabion and the sequences are provided in Supplementary Table S2, as described before^{17 (link)}. 20 µl PCR products were subsequently immobilized to 5 µl Streptavidin Sepharose High Performance Bead (GE Healthcare, Piscataway, NJ, USA), and were finally annealed to 1 µl sequencing primer (5 μM) for 2 min at 80 °C. Amplicons were sequenced using PyroMark Gold Q96 Reagents (Qiagen) on PyroMark Q96 ID System (Qiagen, Hilden, Germany) and analyzed with PyroMark Q CpG software (Qiagen). The relevant sequences are depicted for the five relevant genomic regions in Supplementary Fig. S4. The 15 CpG model for pyrosequencing data, which was trained by lasso regression with the lambda parameter chosen by cross-fold validation, has been described before^{17 (link)} and is provided in Supplementary Table S3.