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The increase in methylation could also be attributed to the release of 5-methylcytosine into the genome. When the dynamic release of 5-methylcytosine was observed using amplification of the endogenous marker 18S rRNA in individual samples, this displayed a similar trend to that of MeDIP-seq (Figure 3B). To confirm this, bisulfite-converted DNA was treated with the CG-specific conversion reagent EZ DNA methylation-direct kit (Zymo Research). Bisulfite-converted 5mC was visualised as a black band on a PCR product, as the M.Rhizobia b-Nseq primer binds to methylated cytosines. PCR products were loaded on a 2% agarose gel, with a visible difference between the bisulfite treated sample, which produced a black band and the control, which did not (Figure 3C). This data suggest that the end of time 2012 peter mettler torrent sequence enrichment for methylation is through the production of 5-methylcytosine during the infection process.
In order to validate this, a RT-qPCR was used to compare MeDIP-seq results for the time-course experiment against the RT-qPCR results for the dynamic release of 5-methylcytosine (Figure 3D). MeDIP-seq displayed an increase in the fraction of unique reads with a methylated cytosine over-represented at the 35S promoter region (Figure 3E, left panel), corresponding to the increase in methylation seen by the bisulfite time course (Figure 3A). To confirm this, bisulfite-treated RNA was treated with the CG-specific conversion reagent and RT-qPCR compared against a mock-treated sample. The CGI methylation assay revealed a significant increase in the proportion of CG-specific amplicons in the treated samples over time (Figure 3E, right panel). This CG-specific assay is therefore able to quantify the increase in DNA methylation as the plant defence response unfolds. d2c66b5586
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