5mC quantification by mass spectroscopy (Assay 1)

The analysis of the percent of 5mC per total nucleotides, by LC-MS/MS, was conducted according to the detailed procedure described before [³⁵]. Briefly, macrophage DNA samples were hydrolyzed to nucleosides by a combination of three enzymes: Nuclease S1, antarthicphosphetase and phosphodiesterase. The chromatographic separations were performed on an Acquity UPLC system (Waters) using Xselect HSS T3 column. Nucleosides were eluted with a gradient of 0.1% formic acid in water and 0.1% formic acid in acetonitrile and directed into a Xevo TQD triple quadrupole mass spectrometer (Waters). The measurements of targeted nucleosides were conducted in a positive ion mode by multiple reaction monitoring (MRM). The relative content of 5mC in each sample was calculated from the MRM peak area of 2ʹ-deoxy-5ʹ-methylcytidine (5mdC) divided by the sum of peak areas of all other nucleosides. A calibration curve was generated by measuring synthetic DNA standards and the sample 5mC content was assessed by interpolation. The measurement error was calculated from the standard error of the linear fit estimate (^{Figure 1(a)}).

Mass spectrometry and optical imaging techniques to quantify 5hmC and 5mC levels of DNA: (a) Assay 1: An MRM chromatogram of nucleosides obtained by hydrolysis of gDNA. (b) Assay 2: Schematic representation of the two-step 5hmC labelling reaction. First, T4 β-GT enzymatic glucosylation of 5hmC with UDP-6-N3-Glu is performed. Next, click reaction between the N3 group and the fluorescently labelled alkyne DBCO-Cy5 is performed. This two-step reaction results in fluorescently labelled 5hmC. (c) Assay 3: Schematic representation of M.TaqI mediated labelling of non-methylated cytosines: Top: M.TaqI catalyzes the transfer of a TAMRA fluorophore from the cofactor AdoYnTAMRA onto the adenine residue that lies in its TCGA recognition site. Bottom: If the cytosine residue that lies within M.TaqI’s recognition site is methylated, the reaction is blocked. (d) Multiple fluorescence microscopy images are obtained per sample, showing both YOYO-1 labelling of the entire DNA molecule (green) and the epigenetic-labels (red dots). In-house developed user interface of the image processing software, where the length of the DNA molecules is measured (green) and colocalized epigenetic-labels (red dots) are detected and subsequently quantified relative to DNA length. Output of the statistics tool, showing the total length of DNA (in bps) and the percentage of epigenetic-labels relative to DNA. Illustrative bar graph comparison of the number of epigenetic labels between two conditions.