Zymo methylation kit

Genome-wide DNA methylation in adipose tissue was analyzed with Illumina Infinium HumanMethylation450k array BeadChips. The array contains 485,577 cytosine probes covering 21,231 (99%) RefSeq genes83 (link). A Zymo Methylation Kit (D5001-D5002, Zymo Research) was used to convert genomic DNA to the bisulfite-modified DNA. Briefly, gDNA (500 ng) of high quality was fragmented and hybridized on the BeadChip, and the intensities of the signal were measured with a HiScanQ scanner (Illumina). The methylation values for each CpG site are presented as a β-value ranging from 0 (unmethylated) to 100% (completely methylated).
The bioinformatics analyses were performed as described21 (link)53 (link). In brief, Y chromosome probes, rs-probe, and probes with average detection p-value > 0.01 were removed. After quality control and filtering, methylation data was obtained in 483,317 CpG sites. Beta-values were converted to M-values (M = log2 (β/(1 − β), which were used for all data analyses. Data were then quantile normalized and batch corrected with COMBAT84 (link). A linear regression model was used to identify differences in DNA methylation between women with PCOS and controls. To improve interpretation, after all the preprocessing steps, data were reconverted to beta-values, which are presented in tables and figures.

DNA was isolated from tissue samples using the Qiagen DNeasy Blood and Tissue Kit (Qiagen Ltd, UK, 69506) and 600 ng was bisulphite converted using the Zymo Methylation Kit (Zymo Research Inc, USA, D5004/8). Genome-wide methylation analysis was performed using the Illumina Infinium Methylation 27 K or 450 K beadchip (Illumina Inc., USA, WG-311-1201 and WG-314-1003). Analysis and quality control were performed as previously described [19 (link), 27 (link), 28 (link)].

Genome-wide DNA methylation in subcutaneous adipose tissue in women with PCOS was analyzed with Illumina Infinium HumanMethylation450k array BeadChips (Illumina, San Diego,CA, USA). The array contains 485,577 cytosine probes covering 21,231 (99%) RefSeq genes⁶⁸ . A Zymo Methylation Kit (D5001-D5002, Zymo Research, Irvine, CA, USA) was used to convert genomic DNA to the bisulfite-modified DNA. Briefly, gDNA (500 ng) of high quality was fragmented and hybridized on the BeadChip, and the intensities of the signals were measured with a HiScanQ scanner (Illumina, San Diego, CA, USA). The methylation values for each CpG site are presented as a β-value ranging from 0 (unmethylated) to 100% (completely methylated).
The bioinformatics analyses were performed as described^{21 (link),69 (link)} at the Department of Clinical Sciences at Lund University in Malmö. In brief, Y chromosome probes, rs–probe, and probes with average detection P–value >0.01 were removed. After quality control and filtering, methylation data were obtained for 483,317 CpG sites. Beta-values were converted to M–values (M = log2 (β/ (1 − β), which were used for all data analyses. Data were then quantile normalized and batch corrected with COMBAT^{70 (link)}. To improve the interpretation, after all the preprocessing steps, data were reconverted to beta-values, which are presented in tables and figures.