Dna degradase plus

Untreated or BS-treated genomic DNA (0.3–1 μg) was digested with a DNA Degradase Plus™ (Zymo Research) for 3 h at 37 °C according to the manufacturer’s instructions. Approximately 50–100 pg per sample were analysed by LC-MS/MS on a Thermo Q-Exactive mass spectrometer coupled to a Proxeon nanoLC. Three replicates of each sample were analysed and the amounts of C, 5mC, 5hmC and U and T were quantified relative to external standards. Recovery of BS-treated genomic DNA for the different BS conversion protocols and clean-up procedures was assessed in the same way as for the M13 fragments, but quantified with LC-MS.

The general protocol is described in Tsuji et al. (2014). Nucleoside quantification was determined using tandem mass spectrometry (MS/MS) coupled with LC (LC‐MS/MS). Genomic DNA was digested with DNA Degradase Plus (ZYMO RESEARCH, Irvine, CA) per the manufacturer protocol. LC separation was performed on a dC18 2.1 × 100 mm column at a flow rate of 0.2 mL/min. The mobile phase was 15% CH₃OH, 85% H₂O with 1% formic acid, and 10 mmol/L ammonium formate. The injection volume was 15 μL. A Waters/Micromass Quattro Micro mass spectrometer was used for the detection of nucleosides. Electrospray ionization in positive ion mode was used to generate ions. Methylation levels are reported as [5 mdC]/[dG], ratio.
Data from 20 trees for each population were analyzed using SPSS 20 for Windows, with all values being log₁₀ transformed to achieve a normal distribution. ANOVA, followed by Tukey's HSD multiple comparison analysis, was performed to determine the significant differences in metal content and global cytosine methylation (P ≤ 0.05) among populations.

Mass spectrometry of nucleosides was performed as previously described (Ficz et al., 2013 (link), Kroeze et al., 2014 (link)). Briefly, 150–1,000 ng genomic DNA was digested using DNA Degradase Plus (Zymo Research) according to the manufacturer’s instructions and analyzed by liquid chromatography-tandem mass spectrometry.

Genomic DNA from both female and male KSR- and FBS-iPSCs, TX 2i ESCs and female and male reprogramming intermediates/non-reprogramming intermediates (day12 and 24) was digested using DNA Degradase Plus (Zymo Research, Cat# E2020) according to the manufacturer’s instructions. Nucleosides were analysed by LC-MS/MS on a Q-Exactive mass spectrometer (Thermo Scientific) fitted with a nanoelectrospray ion-source (Proxeon). All samples and standards had a heavy isotope-labelled nucleoside mix added prior to mass spectral analysis (2′-deoxycytidine-¹³C₁, ¹⁵N₂ (Santa Cruz, Cat# SC-214045), 5-(methyl−²H₃)-2′-deoxycytidine (Santa Cruz, Cat# SC-217100), 5-(hydroxymethyl)−2′-deoxycytidine-²H₃ (Toronto Research Chemicals, Cat# H946632). MS2 data for 5hmC, 5mC and C were acquired with both the endogenous and corresponding heavy-labelled nucleoside parent ions simultaneously selected for fragmentation using a 5 Th isolation window with a 1.5 Th offset. Parent ions were fragmented by Higher-energy Collisional Dissociation (HCD) with a relative collision energy of 10%, and a resolution setting of 70,000 for MS2 spectra. Peak areas from extracted ion chromatograms of the relevant fragment ions, relative to their corresponding heavy isotope-labelled internal standards, were quantified against a six-point serial 2-fold dilution calibration curve, with triplicate runs for all samples and standards.