Methods and kits for genome-wide methylation of GpC sites and genome-wide determination of chromatin structure
Disclosed are methods and kits for genome-wide methylation of GpC sites and for genome-wide chromatin structural determination. Specifically, the methods and kits of the present invention make possible the simultaneous determination of endogenous DNA methylation state and chromatin architecture across the entire genome.
1. A method for genome-wide methylation-sensitive chromatin structure determination comprising:
providing eukaryotic cells with nuclei comprised of chromatin, wherein the chromatin is comprised of nucleosomes having DNA associated with histones;
extracting the nuclei of the cells;
methylating GpC sites of the chromatin by incubating a combination of the nuclei, sucrose and a GpC methylating reagent comprising an effective amount of a GPC methyltransferase and a methyl transfer agent, wherein the GpC methyltransferase is M. CviPI, wherein the concentration of M. CviPI is between about 50 and 500 U in a volume of about 0.5 mL, wherein the concentration of nuclei is about 250,000 nuclei in a volume of about 0.5 mL wherein the solution comprising M. CviPI and nuclei further comprises about 32 mM of the methyl transfer agent SAM, about 1M sucrose and CviPI buffer, wherein the solution is incubated at about 37° C. for about 15 minutes, and wherein the effective amount of the GpC methyltransferase and the methyl transfer agent is an amount effective to methylate GpC sites of chromatin not associated with nucleosomes without substantially methylating GpC sites of chromatin associated with nucleosomes;
purifying the DNA;
bisulfite converting the DNA;
sequencing the DNA; and
determining the endogenous methylation state of the DNA and the GpC sites associated with the nucleosomes.
2. The method of claim 1 , wherein the step of extracting the nuclei comprises a step of lysing the cells to lyse the cytoplasmic membrane of the cell.
3. The method of claim 1 , wherein the effective amount of the GPC methyltransferase and the methyl transfer agent is an amount effective to methylate at least 90% of the GpC sites of the chromatin not associated with nucleosomes.
4. The method of claim 3 , wherein the effective amount of the GPC methyltransferase and the methyl transfer agent is an amount effective to methylate at least 99% of the GpC sites of the chromatin not associated with nucleosomes.
5. The method of claim 3 , wherein the effective amount of the GPC methyltransferase and the methyl transfer agent is an amount effective to methylate less than 10% of the GpC sites of the chromatin associated with nucleosomes.
6. The method of claim 1 , wherein the amount of the GpC methyltransferase is about 100 U.
7. The method of claim 1 , wherein the amount of the GpC methyltransferase is added in more than one aliquot.
8. The method of claim 7 , wherein the more than one aliquot comprises a first aliquot of 200 U and a second aliquot of 100 U.