Ez dna methylation kit

The EZ DNA methylation kit (ZYMO, cat. no. D5001) was used to convert 500 ng sonicated sperm DNA according to the manufacturer’s instructions. In total, 2,000 FACS-sorted male foetal germ cells were lysed with proteinase K solution and converted with EZ DNA methylation kit (ZYMO, cat. no. D5001) according to the manufacturer’s instructions.

DNA was extracted from CB samples or cells (HCT116 and SHSY5Y) by using the QiAMP DNA Blood Mini kit (Qiagen), according to the manufacturer’s instructions. DNA extracted from 41 FA and 45 placebo CB samples and from cells was assessed for quality control by NanoDrop 2000 spectrophotometer (Labtech International, Ringmer, UK) and by agarose gel electrophoresis. DNA from CB samples and HCT116 and DKO cells was quantified by using Quant-IT PicoGreen dsDNA Assay Kit (Invitrogen, Paisley, UK). The DNA at a concentration of 50 ng/μl was sent to Cambridge Genomic Services (Cambridge, UK), performed bisulphite conversion on the DNA in-house using the EZ DNA Methylation Kit (Zymo Research, California, USA) before hybridization to the Infinium Human Methylation EPIC BeadChip Array and scanning with the Illumina iScan according to manufacturer’s instructions (Illumina, Chesterford, UK). The genomic DNA from SHSY5Y cells was bisulphite-converted in-house using the EZ DNA Methylation Kit (Zymo Research, California, USA).

Estimation of DNA methylation profile was performed in a set of 73 EOC patients. At first, bisulfite conversion of 500 ng DNA was done using EZ DNA MethylationTM Kit (Zymo Research, Irvine, CA, USA) according to the manufacturer´s manual. Estimation of genome-wide DNA methylation level for more than 850,000 methylation sites across the genome were done by Infinium MethylationEPIC BeadChip microarray (Illumina Inc.) according to the manufacturer´s recommendations. Microarray was scanned by iSCAN System (Illumina Inc.).

DNA samples at birth were obtained from cord blood, drawn from the umbilical cord upon delivery in accordance with standard procedures. DNA methylation analysis of these samples was performed using the Illumina Infinium HumanMethylation450K BeadChip assay [19 (link)]. All DNA methylation wet-lab and pre-processing analyses were performed at the University of Bristol as part of the ARIES project. Following extraction, DNA was bisulfite-converted using the Zymo EZ DNA MethylationTM kit (Zymo, Irvine, CA). The Illumina 450 K array was used to quantify DNA methylation at over 485,000 CpG sites across the genome. The arrays were then scanned using an Illumina iScan and initial quality review was assessed using GenomeStudio. Samples then underwent a number of further quality control processes. For each sample, the estimated level of DNA methylation at each CpG site was reported as a beta value (β), ranging from 0 (no cytosine methylation) to 1 (complete cytosine methylation) [18 (link)].

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.