DNA methylation and nucleosome positioning work together to generate chromatin 
structures that regulate gene expression. Nucleosomes are typically mapped using 
nuclease digestion requiring significant amounts of material and varying enzyme 
concentrations. We have developed a method (NOMe-seq) that uses a GpC 
methyltransferase (M.CviPI) and next generation sequencing to generate a high 
resolution footprint of nucleosome positioning genome-wide using less than 1 
million cells while retaining endogenous DNA methylation information from the 
same DNA strand. Using a novel bioinformatics pipeline, we show a striking 
anti-correlation between nucleosome occupancy and DNA methylation at CTCF 
regions that is not present at promoters. We further show that the extent of 
nucleosome depletion at promoters is directly correlated to expression level and 
can accommodate multiple nucleosomes and provide genome-wide evidence that 
expressed non-CpG island promoters are nucleosome-depleted. Importantly, 
NOMe-seq obtains DNA methylation and nucleosome positioning information from the 
same DNA molecule, giving the first genome-wide DNA methylation and nucleosome 
positioning correlation at the single molecule, and thus, single cell level, 
that can be used to monitor disease progression and response to therapy.