The molecular mechanism for ssRNA+dsDNA triple helix structure formation on chromatin are unclear. We analyzed the triplex-nucleosomes complex fomration in vitro and in vivo, and show that triplexes are stabilized by the nucleosomes. We developed a method to monitor nucleosome bound RNA triplexes in vivo, revealing that RNA binding maintained the nucleosomes on an accessible structure supporting a gene activating role of nucleosome-triplex complexes in cells. Overall design: DNAse I digestion of chromatin to obtain mono-nucleosome from HeLa nuclei purified using the REAP method.