Metazoan promoters are highly enriched in secondary structure forming motifs, among which G-quadruplexes (G4s). We describe G4access, a novel approach to isolate and sequence G4s when associated to open chromatin, based on their resistance to Micrococcal nuclease. G4access is an antibody- and crosslink-independent procedure that allows for high enrichment of predicted G4s (PG4s) motifs, most of which can be confirmed in vitro. Using this technique in several human and mouse cell lines, we were able to show a cell-specific enrichment that both relates to apparent nucleosome depletion and transcription at promoters. G4access allows scoring for PG4 pattern variation linked to nucleosome positioning changes that occur following treatment with a G4 ligand. It also reveals an unexpected role of G4s in hybrid mouse ES cells in the context of imprinting control regions, in which we propose that they will hallmark allelic active loci. Finally, we extended our procedure to non-mammalian species showing less annotated G4s in their genome. We found a decreased but still substantial G4 enrichment and confirmed their association to open and transcriptionally active regions of the yeast genome. Overall, our study not only provides a novel tool for studying G4 forming sequences in the cellular context but also indicates their essential roles as promoter elements, in chromatin opening and nucleosome positioning. Overall design: We developed G4access, a novel, efficient and Ab and crosslinking-independent method coupled to high-throughput sequencing, that enriches for G4 forming sequences associated to open chromatin in cells. Taking advantage of the sequence preference of the Micrococcal nuclease. G4access signals were then compared to RNA Polymerase II signals upon KM05283 and Tryptolid (Two transcription inhibitors) and Pyridostatin (a G4 ligand).