DNA extraction, whole-genome methylation profiling and bisulfite pyrosequencing
DNA methylation was analyzed with Infinium HumanMethylation450 (HM450) BeadChips (Illumina Inc.). This array provides genomic coverage of a total of 21 231 UCSC RefGenes (including the 5′ and 3′ untranslated regions), 26 658 CpG islands (96%), 59 916 DNAse hypersensitive sites and 80 538 informatically predicted enhancers (). Total DNA was extracted with an adenosine triphosphate (ATP) Genomic DNA MiniKit (ATP Biotech), following the manufacturer's instructions. Quantification and integrity were measured with a Qubit 2.0 Fluorometer (Life Technologies) and 1x Tris Acetate-EDTA-agarose gels, respectively. Starting with 500 ng of good quality total DNA, unmethylated cytosines were converted to uracils using an EZ DNA Methylation Kit (Zymo Research). Subsequently, whole genome methylation profiles were characterized by amplification of converted DNA and hybridization on Infinium HumanMethylation450 (HM450) BeadChips (Illumina Inc.) following Illumina's Infinium HD assay methylation protocol. Datasets were generated from two biological replicates per cell type, which were obtained from a pool of three individuals. Samples were pooled using identical amounts of DNA per donor. Raw data were decoded with GenomeStudio software (Illumina Inc.) to obtain a Final Report (sample probe profile). DNA was extracted from two independent sample sets (one sample at each differentiation step from two different donors) for bisulfite pyrosequencing. Sodium bisulfite modification of 500 ng DNA was performed with the EZ DNA methylation kit (D5002, Zymo Research). Bisulfite pyrosequencing was performed with the PyroMark Q24 reagents (Qiagen), following the manufacturers’ instructions. The PyroMark Assay Design tool (v. 2.0.01.15) was used to obtain pyrosequencing oligonucleotides (Supplementary Table S2). After PCR amplification of the region of interest, methylation levels were quantified using the PyroMark Q24 system (Biotage). Full paper
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