Supplementary Figure 8: CMS IP in MD3d critical period mice.
From: Experience-dependent DNA methylation regulates plasticity in the developing visual cortex
a) CMS technique flow chart. b) Using an anti-CMS antibody to perform the immunoprecipitation of bisulfite-converted DNA we confirmed the results obtained with hMeDIP. MD3d induced a significant decrease in DNA hydroxylmethylation on CRE1, CRE2-3, BDNFex-4b/c, in the visual cortex contralateral to the deprived eye with respect to the ipsilateral cortex (contra ctx vs ipsi ctx, paired t-test, CRE1 n = 5, t = 3.49, P = 0.025; CRE2-3 n = 5, t = 3.76, P = 0.02; BDNFex4-b/c n = 5 t = 3.43, P = 0.03). Error bars represent SEM. CMS-IP has been shown to perform equal or better than glucosylation, periodate oxidation and biotinylation (GLIB) technique or hMeDIP (Pastor, W. A., Huang, Y., Henderson, H. R., Agarwal, S. & Rao, A. The GLIB technique for genome-wide mapping of 5-hydroxymethylcytosine. Nat Protoc 7, 1909-1917, (2012); Huang, Y., Pastor, W. A., Zepeda-MarTnez, J.A. & Rao, A. The anti-CMS technique for genome-wide mapping of 5-hydroxymethylcytosine. Nat Protoc 7, 1897-1908, (2012). The anti-CMS antiserum was generously provided by Dr. Nancy Huang and Dr. Anjana Rao. Since CMS-IP requires bisulfite conversion, to assess the abundance of the DNA loci of interest in the immunoprecipitate we designed new primers insensitive to the methylation status of the complementary DNA sequences. BDNFex-4b and BDNFex-4c could be evaluated with a single primer set designated BDNFex-4b/c.
