Zymo dna methylation gold kit

Genomic DNA was isolated from tissue samples and cultured cells using a Promega DNA purification kit (Promega Corporation, Madison, WI, USA), in accordance with the manufacturer’s protocol. After the purity and concentration of DNA was quantified and assessed, methylated residues were modified using a ZYMO DNA Methylation-Gold kit (Zymo Research Corp., Irvine, CA, USA) to differentiate methylated CpGs from unmethylated CpGs. Using this treatment, unmethylated cytosines were converted into uracil, whereas methylated cytosine remains as cytosine. Subsequently, MS-PCR was performed using the reaction mixtures described in Table II. The PCR conditions were as follows: One cycle of denaturation at 95°C for 5 min, followed by 35 cycles of 95°C for 30 sec, 64 or 58°C (methylated or unmethylated) for 50 sec, and 72°C for 30 sec. Primer sequences for the unmethylated reaction were as follows: Forward, 5′-TAGGATTTGTTTTGTGTATG-3′, and reverse, 5′-ACCACATCACCCATTAACCACA-3′ (94 bp), whereas for the methylated reaction, the following primers were used: Forward, 5′-TAGGATTCGTTTCGCGTACG-3′, and reverse, 5′-ACCGCGTCGCCCATTAACCGCG-3′ (94 bp).

Bisulfite sequencing PCR (BSP) was used as a quantitatively analyzed method for detecting the methylation rate of the RNF180 promoter area. The gene sequence for this study was obtained from Genebank. The MethylKIT package (http://www.bioconductor.org/packages/release/bioc/html/methylKit.html) of R software was used to analyze methylation data, all CpG sites and transcription initiation site (TSS). Genomic DNA sodium bisulfite modification was performed by Zymo DNA Methylation-Gold kit (Cat. Nos. D5005 or D5006, Zymo Research, US) according to the manufacturers’ instructions. Polymerase chain reaction (PCR) was performed by the enzyme TaKaRa Taq™ Hot Start Version (Code No. R007A Takara, Japan). The primers were as follows: F:5′-GTGGTTTTGGTAAGGGGATGAT-3′; R: 5′-AACAACCAAACTCTAAAAACTC-3′.11 (link) The condition was as follows: 94°C for 3 mins for initial denaturation, run 45 cycles (94°C 20 s, 58.5°C 30 s, 72°C 45 s); after the end of the cycles followed by an extension 10 mins at 72°C, a final termination at 4°C. PCR purification products were used for forward sequence analysis. Chromas version 2.6.6 (Technelysium Pty Ltd., Australia) was used to analyze sequencing results. According to the theory of bisulfite modification, methylation rate calculation formula was as follows: Meth%=C/(C+T)*100%.12 (link)

400 ng of genomic DNA isolated from ES cells was bisulfite-converted using a Zymo DNA Methylation Gold Kit (Zymo Research) and then subjected to PCR to amplify target regions using Phusion U Hot Start polymerase (Thermo Fisher Scientific). Both procedures were performed according to manufacturer's instructions. Obtained PCR products were purified using a GeneJET PCR purification kit (Thermo Fisher Scientific) and cloned into the pUC19 vector. Sixteen clones of each sample were randomly selected for Sanger sequencing. CpG methylation status of bisulfite sequencing data was evaluated using BISMA tool (Rohde et al., 2010 (link)). All primers are listed in Table S2.