Zymo ez dna methylation kit

Total DNA from paraffin-embedded tissues, cell lines and EECs were isolated using specific kits (Targene Medical, Wuhu, China) according to the manufacturer’s instructions. Zymo EZ DNA Methylation Kit (Zymo Research, Orange County, California, USA) was used for bisulfite conversion.

The Illumina HumanMethylation450 BeadChip^{62 (link)} was used to assay 146 HB and 11 non-cancerous liver samples. This platform included probes for more than 480,000 CpG sites, spanning 99% of RefSeq genes. Genomic DNA (500 ng) for each sample was treated with sodium bisulfite　and recovered using the Zymo EZ DNA methylation kit (Zymo Research, Irvine, CA), according to the manufacturer’s specifications. The raw signal intensity for methylated and unmethylated DNA was measured using a BeadArray Scanner (Illumina). After color-bias correction, background subtraction of the signal intensities, and inter-array normalization on Genome Studio (Illumina), the raw methylation value for each CpG was defined as β = M/(M + U + 100), where M and U were the intensities of methylated and unmethylated probes, respectively. We then carried out a hierarchical clustering analysis using Cluster 3.0^{63 (link)} with Euclidean distance and complete linkage. Computational estimation of non-tumor cell fractions was performed using InfiniumPurify software^{64 (link)} with informative differentially methylated CpG sites for hepatocellular carcinoma (LIHC).

The DNA methylation data set has been deposited in Gene Expression Omnibus (GSE64495).
Blood samples were drawn sequentially on a given day from all members of the same family into anticoagulant (EDTA) containing tubes, immediately frozen on dry ice and shipped overnight to the processing center. Tubes were coded to prevent bias in subsequent analysis. Genomic DNA was extracted and purified using the RecoverEase DNA Isolation Kit (Agilent Technologies, La Jolla, CA, USA). Bisulfite conversion using the Zymo EZ DNA Methylation Kit (ZymoResearch, Orange, CA, USA) as well as sub-sequent hybridization of the Human Methylation450K Bead Chip (Illumina, SanDiego, CA), and scanning (iScan, Illumina) were performed according to the manufacturers protocols by applying standard settings. DNA methylation levels (β values) were determined by calculating the ratio of intensities between methylated (signal A) and un-methylated (signal B) alleles. Specifically, the β value was calculated from the intensity of the methylated (M corresponding to signal A) and un-methylated (U corresponding to signal B) alleles, as the ratio of fluorescent signals β = Max(M,0)/[Max(M,0)+Max(U,0)+100]. Thus, β values ranged from 0 (completely un-methylated) to 1 (completely methylated). We used background corrected beta values that result from the BeadStudio software (version 3.2).