Because of its profound influence on DNA accessibility for protein binding and thus on the regulation of diverse biological processes, nucleosome positioning has been studied for many years. In the past decade, high-throughput sequencing technologies have opened new perspectives in this research field by allowing the study of nucleosome positioning and occupancy on a genome-wide scale, therefore providing understanding on important aspects of chromatin packaging, as well as on various chromatin-template processes like transcription. In this chapter, we provide the protocol of MNase sequencing for the genome-wide mapping of nucleosomes using MNase to generate mononucleosomal DNA fragments and next-generation sequencing technology to identify their individual location.