Epijet 5 hmc and 5 mc analysis kit

Aliquots (100–150 μl) of phage suspensions (10¹¹–10¹² PFU/ml) were subjected to phenol/chloroform extraction and ethanol precipitation. Isolated phage DNA was used for restriction analysis with Eco32I, MboI, EcoRII, SalI and Csp6I restriction endonucleases (Thermo Fisher Scientific) according to supplier’s recommendations. In vitro DNA glycosylation tests were performed in the Epi Buffer using T4 phage β-glucosyltransferase (T4 BGT) and UDP-glucose from the EpiJET 5-hmC and 5-mC Analysis Kit (Thermo Fisher Scientific). DNA fragments were separated by electrophoresis in a 0.8% agarose gels stained with ethidium bromide.

The absolute level of 5-hmC in genomic DNA, previously extracted from 34 glioma tissues, was estimated with the EpiJET 5-hmC and 5-mC Analysis Kit (ThermoFisher Scientific). Briefly, 100 ng of DNA was glucosylated by T4 phage β-glucosyltransferase (T4 BGT), followed by subsequent digestion with Epi MspI and Epi HpaII enzymes. Then the percentage of cytosine modifications within CCGG sites was determined by quantitative real-time PCR (qRT-PCR) with a primer pair flanking recognition sequence. Primer sequences were as follows:
primer1_forward 5′-CTGTCATGGTGACAAAGGCATC-3′,
primer2_reverse 5′-CAGGATTTCTCTATTATGAAGACCTTG-3′.
The experiment was run in triplicate and the amount of 5-hmC was calculated as a percentage based on controls included in the kit.

The level of 5-mC and 5-hmC was assessed using Thermo Scientific EpiJET 5-hmC and 5-mC Analysis Kit (Thermo Scientific™, ThermoFisher Scientific, Waltham, MA, USA). According to the manufacturer’s instructions, each glucosylation reaction used 1 µg of DNA. Digestion with the restriction enzymes T4 BGT, Epi MspI and Epi HpaII was then performed according to the manufacturer’s protocol. Pre-prepared samples were subsequently analyzed using qPCR with specific primers flanking selected CpG sites (Table S1) and Maxima SYBR Green/ROX qPCR Master Mix (Thermo Scientific™, ThermoFisher Scientific, Waltham, MA, USA). The qPCR was performed using a CFX Connect Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). For the qPCR reaction, 1 µL of digested DNA was used as a template, 10 µL of PCR Master mix, 0.3 µM of forward and reverse primers and the appropriate amount of nuclease-free water. The total volume of the reaction mixture was 20 µL. Each trial was performed in two independent replicates. The qPCR conditions are shown in Table S2.