TY  - JOUR
TI  - Preparation of Chromatin Fragments From Human Cells for Cryo-EM Analysis
AU  - Hatazawa, Suguru 
AU  - Takizawa, Yoshimasa 
AU  - Kurumizaka, Hitoshi 
VL  - 15
IS  - 20
PY  - 2025
DA  - 2025/10/20
SP  - e5472
C1  - Bio-protocol 2025;15:e5472
DO  - 10.21769/BioProtoc.5472
UR  - https://doi.org/10.21769/BioProtoc.5472
AB  - Eukaryotic genomic DNA is packaged into chromatin, which plays a critical role in regulating gene expression by dynamically modulating its higher-order structure. While in vitro reconstitution approaches have offered valuable insights into chromatin organization, they often fail to fully capture the native structural context found within cells. To overcome this limitation, we present a protocol for isolating native chromatin fragments from human cells for cryo-electron microscopy (cryo-EM) analysis. In this method, chromatin from formaldehyde-crosslinked human HeLa S3 nuclei is digested with micrococcal nuclease (MNase) to generate mono- and poly-nucleosome fragments. These fragments are subsequently fractionated by sucrose-gradient ultracentrifugation and prepared for cryo-EM. The resulting chromatin fragments retain native-like nucleosome–nucleosome interactions, facilitating structural analyses of chromatin organization under near-physiological conditions.
KW  - Native chromatin
KW  - Native nucleosome
KW  - Human cells
KW  - Cryo-electron microscopy
KW  - Cryo-electron tomography
JF  - Bio-protocol
SN  - 2331-8325
PB  - Bio-protocol LLC.
BIO101  - False