Epimark 5 hmc and 5 mc analysis kit

Total DNA methylation (%5mC) and hydroxymethylation (%5hmC) were assessed using “MethylFlash” global DNA methylation ELISA kit and “MethylFlash” global DNA hydroxymethylation ELISA kit, accordingly (EpiGentek).
Sequence specific DNA methylation (%5mC) and hydroxymethylation (%5hmC) was assessed using the “EpiMark 5-hmC and 5-mC Analysis kit” (NEB, Ipswich, MA) according to the manufacturer’s protocol. Briefly, DNA from day 10 post-hatch anterior hypothalamus, was incubated with T4 β-glucosyltransferase (T4-BGT, which adds a glucose mark on 5hmC, creating 5ghmC), 37°C, overnight. T4-BGT⁺ and T4-BGT^– samples were than incubated with MspI (cleaves DNA at CCGG, unless the middle CG is 5ghmC) or HpaII (cleaves DNA at CCGG unless the middle CG is modified to 5mC, 5hmC, or 5ghmC) restriction enzymes, 37°C, 12 h. The restriction products are later incubated with proteinase K, 40°C, 30 min, followed by 10 min of 95°C for proteinase K inactivation. DNA samples were used in qPCR to determine %5hmC and %5mC according to the expression of specific targets.
t1 = expression of T4-BGT⁺, Msp⁺; t3 = expression of T4-BGT⁺, uncut; t4 = expression of T4-BGT^–, Msp⁺; t5 = expression of T4-BGT^–, Hpa⁺; t6 = expression of T4-BGT^–, uncut.
For qPCR we designed primers amplifying regions containing CCGG, which presented increased probability for binding transcription factors via the TFBIND algorithm¹.

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.

DNA glucosylation and restriction endonuclease digestions were performed using the Epimark 5-hmC and 5-mC analysis Kit (NEB, Ipswich, MA) as per the manufacturers instructions. The primer sequences used in this analysis were listed in Additional file 2: Supplementary Table 1. A total of 5ug of genomic DNA was treated with T4 β-glucosyltransferase with and without UDP-Glucose substrate at 37 °C for overnight. Glucosylated DNA was digested with and without MspI and HpaII at 37 °C for overnight. 5hmC levels were quantitatively analysed using Real time Q-PCR with primers designed at peak regions containing GGCC sequence on target genes which were shown to be differentially hydroxymethylated between CLL samples and normal B cells (Additional file 7).