T4 bgt

5hmC was fluorescently labelled in a two-step chemical labelling procedure [22 (link)]. Three hundred nanograms of genomic DNA was mixed with 3 μl of buffer 4 (NEB), 0.5 μl of UDP-6-N₃-Glu (0.3 mM, [20 (link)]), 2 μl of T4 bacteriophage β-glucosyltransferase (T4-BGT, NEB) and ultrapure water to a final volume of 30 μl. The reaction was mixed and incubated overnight at 37 °C. On the following day, 0.15 μl of DBCO-Sulfo-Cy5 (10 mM, Jena Biosciences) was added to the mixture, followed by a second overnight incubation at 37 °C (click reaction). Upon completion of the labelling procedure, residual fluorophore was removed by drop dialysis (0.1 μm, Millipore) against 300 ml of TE buffer for 2–3 h. Alternatively, samples were washed by ethanol precipitation or by magnetic beads (MagVigen DNA Select Kit, NVIGEN). Samples were stored at 4 °C until analyzed.

To analyze and quantitate 5-mC and 5-hmC within a specific locus, we used an EpiMark 5-hmC and 5-mC Analysis Kit (NEB, MA, USA) according to the manufacturer's instructions. In brief, DNA was isolated from embryos at the blastocyst stage (n = 10). DNA was then subjected to T4 Phage β-glucosyltransferase (T4-BGT, NEB) treatment for 18 h. Glycosylated DNA was digested with 40U of HpaII, 100U of MspI or no enzyme (mock digestion) at 37°C for 18 h, which was followed by treatment with Proteinase K (PK) for 30 min at 40°C. The subsequent inactivation of PK was performed at 98°C for 10 min. HpaII- and MspI-resistant fractions were used for real time qRT-PCR with primers that were designed around the MspI (HpaII) site. DNA subjected to the glycosylation treatment that was not completely digested by MspI at the MspI site was considered to contain 5-hmC at this site. The real time qRT-PCR reaction (25 μl) consisted of 2μl of DNA, 12.5μl of SYBR Green master mix (TaKaRa, Japan), 9.5μl of RNase-free water and 0.5μl of both forward and reverse primers (10 pmol) for each gene. The real time qRT-PCR protocol consisted of a denaturing cycle of 10 min at 95°C and 40 cycles of PCR (95°C for 10s, 60°C for 30 sec, 72°C 20 sec). The relative amounts of DNA were analyzed using the 2^−ΔΔCT method.