ALT Figure 1. (A) Classical gel electrophoresis experiments showing mono-, di-, tri-, tetra-, and further multinucleosome bands upon chromatin digestion. (B) The nucleosome repeat length (NRL) is defined as the genomic distance between the centres of two neighbouring nucleosomes.

ALT Figure 2. Nucleosome mapping using MNase-seq versus ATAC-seq. (A) In MNase-seq, nucleosomes in both open and tightly packed genomic regions are accessible to digestion. MNase preferentially cleaves DNA between nucleosomes and digests DNA until it encounters a histone octamer, which provides a footprint of nucleosome-protected DNA regions. (B) Bulk MNase-seq results in averaged maps across millions of cells, effectively capturing all possible nucleosome positioning configurations. (C) Single-cell MNase-seq (scMNase-seq) results in a noisier and sparser signal. The resulting footprints still represent nucleosome-protected regions, but not all nucleosomes are represented. (D) In ATAC-seq, open regions can be accessed by the enzyme Tn5 transposase, which can insert primers in regions free from the binding of nucleosomes and transcription factors (TFs). (E) For open chromatin regions, nucleosome maps can be obtained from ATAC-seq similar to MNase-seq. (F) Closed, tightly packed chromatin...

ALT Figure 5. Molecular mechanisms affecting nucleosome spacing. (A) Linker histones H1 and nonhistone chromatin proteins which compete with H1s and modulate nucleosome spacing through structural and electrostatic mechanisms. (B) Chromatin remodellers actively reposition nucleosomes following context-dependent rules. (C) Cell state-dependent chromatin boundaries formed by CTCF and other structural proteins, as well as associated recruitment of chromatin remodellers which space nucleosomes. (D) Gene activity associated with remodeller action and RNA polymerases transcribing through the nucleosomes, leading to smaller distances between nucleosomes in regulatory regions and gene bodies. (E) DNA sequence repeats of different types.

ALT Figure 6. Examples of NRL changes in biological systems. (A) Cell differentiation leads to NRL changes between different cell types, e.g. mouse dorsal root ganglia neurons (NRL ∼165 bp) versus cortical astrocytes (NRL ∼183 bp) [175]. Schematic cell shapes are adapted from an image created in BioRender (https://BioRender.com/89trj2t). (B) Paired normal versus tumour breast tissues show NRL shortening in cancer (figure adapted from [36] under the CC BY 4.0 licence (https://creativecommons.org/licenses/by/4.0/)). (C) Nucleosome positioning derived from cfDNA of human volunteers shows NRL increase with age (figure reprinted from [79] under the CC BY 4.0 licence (https://creativecommons.org/licenses/by/4.0/)).