Methylated dna standard kit

Hydroxymethylated DNA immunoprecipitation (hMeDIP) was performed to analyse the 5-hmC enrichment in the gene promoters. Genomic DNA was prepared by QIAamp DNA blood mini kit (Qiagen) and fragmented by sonication to a size range of 200-1000bp confirmed by gel electrophoresis. As a positive control for 5-hmC, we used together with our tested DNA 1μl of hydroxymethylated DNA of APC (adenomatous polyposis coli) promoter from the methylated DNA standard kit (Active Motif, USA). Denatured at 99°C for 10 minutes, 10% of the genomic DNA was collected as input, the rest was incubated with 1μl 5-hmC antibodies (Active Motif) at 4°C overnight. Dynabeads Protein A (Invitrogen / Thermo-Fisher, Switzerland) was applied to capture the hydroxymethylated DNA. Immunoprecipitated (IP) samples were released from the beads by resuspension in digestion buffer with protease K and incubation at 50°C for 2 hours in a shaking heat block at 800rpm. IP and input samples were purified by QIAquick PCR purification kit (Qiagen) and analysed by quantitative Real-time PCR in the Taqman 7900 PCR system (Applied Biosystems) using specific primers for the TNFαpromoter (Table 3). The enrichment was presented as ratio of IP copy number to input copy number.