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Supplemental Figure 5: Global DNA methylation. DNA was extracted using the DNAeasy tissue kit (Qiagen, 69504) from embryos treated with DMSO or MTX. The optional RNAse treatment was performed. Restriction digests were performed using the Epijet DNA methylation kit (Thermo, K1441) which uses the isoschizomers HpaII and MspI which have different sensitivities to CpG methylation. When the internal CpG in the 5’-CCGG-3’ tetranucleotide sequence is methylated, cleavage with HpaII is blocked, but cleavage with MspI is not affected. Therefore, modifications in global methylation levels would be revealed by a change in the ratio of HpaII to MspI. Digest products were run on a 2% agarose gel and no obvious difference was observed between treatments in 6 experiments. The uppermost band was quantified and the ratio of HpaII to MspI also showed no statistical difference. Image published in: Wahl SE et al. (2015) Copyright © 2015. Image reproduced with permission of the Publisher, Elsevier B. V. Larger Image Printer Friendly View

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