Zymo ez dna methylation kittm

Genomic DNA (800 ng) treatment with sodium bisulphite was done with the Zymo EZ DNA Methylation KitTM (Zymo Research, Orange, CA, USA) according to the manufacturer’s procedure, with the alternative incubation conditions recommended when using the Illumina Infinium Methylation Assay. The methylation assay was performed on 4 μl bisulphite-converted genomic DNA at 50 ng/μl according to the Infinium HD Methylation Assay protocol. Bead chip arrays were scanned on Illumina Scan and raw .idat files were generated.

Genomic DNA was extracted as described above. Site-specific CpG methylation was analyzed by means of the Infinium^® HumanMethylation27 bead-array-based technique. This array was developed to assay 27,578 CpG sites selected from over 14,000 genes. The Zymo EZ DNA Methylation KitTM (Zymo Research, Orange, USA) was used to treat 1 μg genomic DNA with sodium bisulfite. This was done according to the manufacturer's procedure, with the alternative incubation conditions recommended when using the Illumina Infinium^® Methylation Assay. The assay was performed on 4 μL converted gDNA at 50 ng/μL concentration, according to the protocol described in the Infinium^® Methylation Assay Manual. Raw Infinium data were filtered by removing low quality data using a detection P-value threshold of 0.05. As the vast majority of Illumina HumanMethylation27 targets are covered by the HumanMethylation450 we used the extended annotation provided by Price et al. [78 (link)] to filter out the Probes containing SNPs (see [11 (link)] for a detailed description). Probes associated to X and Y chromosomes were removed from the analysis. β-values were computed using the formula β-value = M/[U + M] where M and U are the raw “methylated” and “unmethylated” signals, respectively.