Pyromark cpg software 1

Genomic DNA of primary fibroblasts was extracted with the High Pure PCR Template preparation Kit (Roche) according to the manufacturer’s instructions. Bisulfite conversion of 500 ng genomic DNA per sample was performed with the EpiTect Bisulfite Kit (Qiagen) according to the manufacturer’s specifications. Quantification of DNA methylation was carried out by PCR of bisulfite-converted DNA and pyrosequencing using a PyroMark Q96 ID instrument (Qiagen) and PyroMark Gold Q96 reagents (Qiagen). Data were analysed using the PyroMark CpG Software 1.0.11 (Qiagen).

Human genomic DNA was isolated with the DNeasy blood and tissue kit (Qiagen) according to the manufacturer’s instructions and quantified using a NanoDrop 2000 spectrophotometer. 450 ng DNA per sample were used for bisulfite conversion with the Epitect 96 bisulfite kit (Qiagen). PCR and pyrosequencing with primers for the DMRs of SNRPN, LIT, PEG3, H19, MEST and GTL2 as well as LINE1 and Alu elements were performed as described elsewhere [60 (link)]. Pyrosequencing was performed on a PyroMark Q96 ID (Qiagen) with PyroMark Gold Q96 reagents (Qiagen). Data were analysed using the PyroMark CpG Software 1.0.11 (Qiagen).

Genomic DNA isolation from cells and bisulfite pyrosequencing were performed as previously described41 (link). Briefly, Genomic DNA of MEFs, iPSCs and ESCs was extracted with the GentraPuregeneKit (Qiagen) according to the manufacturer’s instructions and quantified using a NanoDrop 2000 spectrophotometer. Bisulfite conversion of 500 ng genomic DNA per sample was performed with the EpiTect Bisulfite Kit (Qiagen) according to the manufacturer’s specifications. Quantification of DNA methylation was carried out by PCR of Bisulfite-converted DNA and pyrosequencing. PCR and sequencing primers for bisulfite pyrosequencing were designed using the Pyrosequencing Assay Design Software 2.0 (Qiagen) and are listed in Supplementary Table 2. For pyrosequencing, a PyroMark Q96 ID instrument (Qiagen) and PyroMark Gold Q96 reagents (Qiagen) were used. Data were analysed using the PyroMark CpG Software 1.0.11 (Qiagen).