Infinium dna methylation platform humanmethylation450 beadchip

ASC-derived iPS (AiPS) and DiPS cells were used. Genomic DNA from AiPS/DiPS and from their corresponding mesenchymal starting cell populations was extracted from approximately 1 × 10⁶ cells using a QIAamp DNA Mini Kit (Qiagen) as per the manufacturer’s instructions. Approximately 1 μg genomic DNA of each sample was bisulphite converted and subsequently processed for profiling with the Illumina Infinium DNA methylation platform (HumanMethylation450 BeadChip) at the Genome Biology Facility at Duke-NUS Medical School, Singapore. The methylation data were subject to quality control and processed in R using the Minfi package (version: 1.18.2) [30 (link)], removing probes with a detection p value greater than 0.05 in more than 25% of all samples, probes which contain common single nucleotide polymorphisms (SNPs), and probes on the sex chromosomes. Differential methylation analysis based on M values was performed using two-way analysis of variance (ANOVA), controlling for matched sample sources and correcting for multiple testing using the Benjamini-Hochberg method. In addition, ingenuity pathway analysis (IPA) was also performed to analyse the set of genes that undergo significant epigenetic changes while transforming from the somatic cell state into the iPS cell state.

Genomic DNA samples (1 ug each) were bisulfite converted using the Zymo EZ DNA methylation kit (Zymo Research, Orange, CA, USA; catalog #D5002) according to the manufacturer’s instructions. All cell lines passed bisulfite conversion quality control and were subsequently processed for the Illumina Infinium DNA methylation platform (HumanMethylation450 BeadChip). A beta value (β) of 0–1.0 was reported for each CpG site (methylation ranging from 0% to 100%, respectively). β values were calculated by subtracting background and taking the ratio of the methylated signal intensity to the sum of both unmethylated (U) and methylated (M) signals: M/(M + U).
The McGill University Genome Quebec Innovation Centre in Montreal performed the Infinium methylation assays in accordance with the manufacturer’s instructions. The assay information is available at http://www.illumina.com. The Infinium array cover 482,421 CpG sites of approximately 28 million sites in the human genome and covers 99% of RefSeq genes, averaging 17 CpG sites per gene. It covers 96% of human CpG islands, with additional coverage in island shores and the regions flanking them.

For extraction of nuclei, cells were trypsinized and centrifuged for 5 min at 200 × g, then washed with ice-cold PBS and resuspended in 1 ml of ice-cold nuclei buffer (10 mM Tris, pH 7.4, 10 mM NaCl, 3 mM MgCl₂, 0.1 mM EDTA and 0.5% NP-40, and protease inhibitors) per 5 × 10⁶ cells and incubated on ice for 5 min. Nuclei were recovered by centrifugation at 900 × g for 3 min and washed in nuclei wash buffer (10 mM Tris, pH 7.4, 10 mM NaCl, 3 mM MgCl₂ and 0.1 mM EDTA containing protease inhibitors). Fresh nuclear preparations (2 × 10⁵ cells) were resuspended in 1×M.SssI reaction buffer (NEB), then treated with 50U of M.SssI (NEB) with 15 μl 10× reaction buffer, 45 μl 1M sucrose, and 0.75 μl S-Adenosyl methionine (SAM) in 150 μl volume. Reactions were quenched by an equal volume of stop solution (20 nM Tris-HCl [pH 7.9], 600 mM NaCl, 1% SDS, 10 mM EDTA and 400 μg/ml proteinase K) and incubated overnight at 55°C (23). DNA was purified by phenol/chloroform extraction and ethanol precipitation. Bisulfite conversion was performed using the EZ DNA methylation kit (Zymo Research). All samples passed bisulfite conversion quality control, and were subsequently processed for the Illumina Infinium DNA methylation platform (Human Methylation 450 Bead Chip). For validation of enzyme treatment and local accessibility, molecules were cloned using the Topo TA Kit (Invitrogen) and sequenced.