On what basis have you selected the speed and time for ultracentrifugation?

Thank you for your questions, and I apologize for the delayed response.

Regarding the ultracentrifugation conditions, we adjusted the sucrose gradient density based on the work of Dacher et al. (Nucleic Acids Research, 2019) to allow for the purification of larger poly-nucleosomes.

Under the current conditions described in our protocol, it is possible to individually separate mono- to tetra-nucleosomes. However, poly-nucleosomes containing more than four nucleosomes cannot be resolved into individual nucleosome units using the present centrifugation parameters.

We have not optimized the centrifugation conditions to achieve finer separation of higher-order chromatin structures. Based on our observations, poly-nucleosome fractions obtained under the current conditions contain a heterogeneous mixture of fiber-like and nucleosome clutch-like assemblies. These findings suggest that while the protocol is efficient for enriching poly-nucleosome populations, further optimization may be required to resolve higher-order chromatin structures.

Suguru Hatazawa