Genome-wide DNA Methylation Profiling

Cells were transfected and/or treated as indicated in figure legends. A total of ~5 × 10⁶ cells were harvested and Genomic DNA extracted as described above. Ten micrograms of total genomic DNA was digested in 200 μl for 16 h with Restriction Endonuclease mix containing 30 U each of Eco RI, Bam HI, Hind III, XbaI, Sal I (Roche Applied Science), phenol/chloroform extracted, ethanol precipitated and resuspended in 50 μl of TE buffer. An aliquot (1/10) of digested DNA was used as input control to determine DNA concentration and digestion efficiency. MeDIP was performed essentially as described32 (link) except that 2 μg of antibody specific for 5 mC (Abcam) were used to precipitate methylated DNA from 5 μg of total genomic DNA.

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Russo G., Landi R., Pezone A., Morano A., Zuchegna C., Romano A., Muller M.T., Gottesman M.E., Porcellini A, & Avvedimento E.V. (2016). DNA damage and Repair Modify DNA methylation and Chromatin Domain of the Targeted Locus: Mechanism of allele methylation polymorphism. Scientific Reports, 6, 33222.

Publication 2016

Eco RI Bam HI Hind III XbaI

Antibody Buffer Cells Chloroform Digestion Eco ri Ethanol Genomic Phenol Restriction endonuclease

Corresponding Organization :

Other organizations : Institute for Experimental Endocrinology and Oncology, University of Naples Federico II, TopoGEN (United States), Columbia University Irving Medical Center

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Variable analysis

independent variables

Transfection and/or treatment of cells as indicated in figure legends

dependent variables

Methylation status of genomic DNA

control variables

Total amount of genomic DNA (5 μg)
Restriction endonuclease digestion (30 U each of EcoRI, BamHI, HindIII, XbaI, SalI)
MeDIP antibody concentration (2 μg of 5mC antibody)

controls

Input control (1/10 of digested DNA) to determine DNA concentration and digestion efficiency

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