Ez dna methylationtm kit

DNA was extracted and processed at the Epigenetics Group, IARC. Specifically, DNA was bisulfite-converted using the Zymo EZ DNA methylation^TM kit (Zymo, Irvine, CA, United States). DNA was then hybridized to Illumina Infinium Human Methylation 450K BeadChip arrays (66) and scanned using the Illumina HiScanSQ system. After background subtraction using Illumina GenomeStudio raw intensity data were submitted to pre-processing, including normalization, using in-house software within the R statistical computing environment. Furthermore, quality control of samples was carried out and failed samples were excluded on the basis of Illumina’s detection p-value greater than 0.01 and bead count lower than 3. For probes using the Infinium II design additional background subtraction and dye bias correction were performed. Methylation levels at each CpG locus were expressed by Beta-values, as defined by the ratio of signal intensity originating from methylated CpGs over the sum of methylated and unmethylated CpGs. Finally, data were trimmed for outliers with values larger than 3 interquartile ranges below the first quartile or above the fourth quartile. CpG sites were annotated using the Bioconductor package IlluminaHumanMethylation450kanno.ilmn12.hg19 in R for the annotation of Illumina’s 450K methylation arrays.

Genomic DNA was isolated from DU145 cells for bisulfite conversion using the EZ DNA Methylation ^TM Kit according to the manufacturer’s protocol (Zymo research, Los Angeles, USA). The DNA was denatured to single-stranded DNA by adding 0.1 N NaOH. After neutralization, the denatured DNA was incubated with whole genome amplification reagent at 37 °C overnight. The DNA fragments were precipitated by adding isopropanol, and precipitated and purified by centrifugation at 4 °C. After air drying, the DNA pellets were re-dissolved in a hybridization buffer reagent. The resuspended DNA was hybridized on the prepared chip in a hybridization oven overnight. The unhybridized and non-specifically hybridized DNA was washed away for subsequent staining and extension. The captured DNA was used as a template, and a single-base extension reaction was performed on the chip. A detectable label group was added to the chip to distinguish DNA methylation. The XC4 reagent was added to the reaction-completed chip, and then dried for 1 h. The processed chips were scanned by a scanner. Because a laser excites the fluorescence of the single-base extension product on the chip, the scanner obtained a high-resolution fluorescence picture. The data were directly imported into the GenomeStudio software for analysis to obtain the methylation data of each sample.

To convert mitochondrial DNA into bisulfate for sequencing, plasma samples from 8 participants were used to isolate platelet mtDNA as described before. Briefly, platelet pellets were obtained by centrifugation of 400 μL of plasma samples at 1,400 × g for 15 min at room temperature. To minimize contamination with nuclear DNA, we added 3 μL of DNase I (Solarbio, China, 10 U/μL) and 7 μL deionized water and incubated the samples for 3 h at 37°C. The samples were treated with 20 μL proteinase K at 50°C for 1 h and centrifuged at 1,000 × for 10 min for purification, and then the supernatants were retained, respectively. According to the protocol provided by the EZ DNA methylation^TM Kit (Zymo Research, CA, United States), the mitochondrial DNA was extracted and transformed into bisulfite-converted mtDNA.

For measuring DNA methylation, DNA was bisulphite-modified (resulting in deamination of unmethylated cytosine to uracil) using an EZ DNA methylation^TM kit (ZYMO Research, Irvine, CA, USA), according to the manufacturer’s protocol, and subjected to quantitative methylation-specific PCR (qMSP), as previously described53 (link). PCR qMSP amplification was performed using the StepOne Real-Time PCR instrument (Applied Biosystems, Carlsbad, CA, USA), with ACTB used to normalize the input DNA. The absolute amount of methylated NR4A3 was deter ed by the threshold PCR cycle number (Ct), for each sample, using a standard curve generated by qMSP of an SssI-treated cloned DNA fragment. The relative percentage of NR4A3 methylation was calculated as the NR4A3 vs ACTB ratio for each sample, divided by the same ratio for SssI-treated sperm DNA (positive DNA methylation control, Millipore, Billerica, MA, USA) and multiplied by 100. Experiments were repeated twice.

The Agena Bioscience EpiTYPER^® system (San Diego, CA, United States) used PCR amplicons of 362 base pairs (bp) for ELOVL2, 191 bp for FHL2 and 344 bp for MIR29B2. Samples analyzed using EpiTYPER^® were bisulfite converted using the EZ DNA Methylation^TM Kit (Zymo Research) using 300 ng of genomic DNA. A detailed description of the EpiTYPER^® workflow has been previously reported (Freire-Aradas et al., 2016 (link), 2018 (link)). Methylation data were obtained using EpiTYPER^® software v.1.2.22 (Agena Bioscience).

A total of 25 CpG sites of the TERT promoter spanning nucleotides (nts) −746 to −445 (positions are given relative to the transcription start site) within nts 1254,147–1253,148 (Figure 1) (38 (link)). The obtained sequence data (accession NC_000005) in the GenBank database (http://www.ncbi.nlm.nih.gov) have been examined with binding sites for myeloid-specific zinc finger protein 2 (MZF2). Bisulfite sequencing was used to assess DNA methylation of the TERT promoter. Genomic DNA was given bisulfite modification with the EZ DNA Methylation TM kit (ZYMO Research, USA). Then, nested PCR was conducted on the methyl-modified DNA with special primers, and the products were sequenced directly. Specific primers for the TERT promoter were as follows: F, 5′- TTTGAGAATTTGTAAAGAGAAATGA-3′; inner R, 5′-AATATAAAAACCCTAAAAACAAATAC-3′; and outer R, 5′-AAAAAAACCATAATATAAAAACCCT-3′ (38 (link)). DNA methylation was calculated from the amplitude of cytosine and thymine within each CpG dinucleotide, C/(C+T), as described by Lewin et al. (40 (link)).

Bisulfite conversion of 500 ng DNA was performed with a EZ DNA Methylation^TM Kit (Zymo Research, Irvine, CA, USA), according to the manufacturer’s protocol. The genome-wide DNA methylation was assessed by the Infinium Human MethylationEPIC BeadChip platform (Illumina, San Diego, CA, USA) following the manufacturer’s instructions. The microarray was scanned by the Illumina iScan system. The obtained data were further processed using the R language [46 ]. Quality control and data normalization were carried out in the minfi package as described previously [47 (link),48 (link)]. Raw data were converted into β values for further analysis [49 (link),50 (link)]. Probes mapped to single nucleotide polymorphism were removed from the analysis [51 (link)]. Differentially methylated probes were defined with |Δβ| > 0.2 (20% difference). The β value is defined as the ratio between methylated versus unmethylated alleles.