Nucleosomes consisting of approximately 146 base pairs (bp) of DNA wrapped around a histone octamer are the fundamental structural units of chromatin in metazoans (1, 2). The translational positioning of nucleosomes along DNA is implicated in profoundly influencing gene expression (3-6). Thus, defining the nucleosome positioning and occupancy is critical to understand the mechanisms of regulation of transcription by chromatin.
Nucleosome structure is resistant to microccocal nuclease (MNase) digestion, leaving a footprint of about 150bp that reflects the position of a nucleosome (7). Therefore, determining the boundaries of these footprints can indicate the positions of nucleosomes in the genome. Since the genomic sequences of most model organisms are already available, sequencing a short tag from DNA at each end of the nucleosome is sufficient to determine its position in the genome. Thus the next generation sequencing techniques are perfectly suited for this purpose (8).
We have generated genome-wide maps of nucleosome positions in both resting and activated human CD4+ T cells by direct sequencing of nucleosome ends using the Illumina Genome Analyzer Platform (MNase-Seq) (9). As the next generation sequencing techniques improve, the capacity and cost of sequencing become lower. For example, one sequencing run on the Illumina Genome Analyzer II can produce 100 to 200 millions of sequencing reads, which is sufficient to reach a 10x coverage for all nucleosomes in the human genome.
We describe two different methods to prepare nucleosome templates used for sequencing. One is digestion of native chromatin and the other is digestion of formaldehyde-crosslinked chromatin by MNase. The native nucleosome protocol works well to reveal stable nucleosome structure and avoid crosslinking of non-histone proteins; the crosslinking protocol may stabilize “unstable” nucleosomes but may also stabilize non-nucleosome structure that is resistant to MNase digestion.