Protocol for integrative analysis of transcription factor-nucleosome interactions using SeEN-seq and cryo-EM structure determination
Kobayashi W, Michael AK, Ruangroengkulrith S, Kümmecke M, Tachibana K. 2025. Protocol for integrative analysis of transcription factor-nucleosome interactions using SeEN-seq and cryo-EM structure determination. STAR Protocols. 7, 104295.
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| Epub ahead of print
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Author
Kobayashi, Wataru;
Michael, Alicia K.ISTA 
;
Ruangroengkulrith, Siwat;
Kümmecke, Maximilian;
Tachibana, Kikuë
;
Ruangroengkulrith, Siwat;
Kümmecke, Maximilian;
Tachibana, KikuëCorresponding author has ISTA affiliation
Department
Abstract
Pioneer transcription factors (TFs) possess the ability to read out DNA motifs embedded within nucleosomes, driving changes in gene expression during cellular differentiation and reprogramming. Here, we present selected engagement on nucleosome sequencing (SeEN-seq), a protocol designed to systematically identify potential TF-binding sites on the nucleosome. We describe steps for nucleosome library assembly, SeEN-seq assay, and cryoelectron microscopy (cryo-EM) sample preparation. This protocol facilitates the preparation of homogeneous pioneer TF-nucleosome complexes for cryo-EM structure determination using single-particle analysis.
For complete details on the use and execution of this protocol, please refer to Michael et al.1
Publishing Year
Date Published
2025-12-26
Journal Title
STAR Protocols
Publisher
Elsevier
Acknowledgement
We thank R.H. Kim, A. Casper, and R. Gautsch for sequencing at the NGS facility (RRID:SCR_025746). K.T. is an Honorary Professor at the Department of Biology, Ludwig-Maximilians-University, Munich, Germany. This study was funded by European Research Council grant ERC-CoG-818556 TotipotentZygotChrom (K.T.), Max Planck Society (K.T.), and ERC Starting Grant “ChromaChrono” 101162145 (A.K.M.).
Volume
7
Article Number
104295
eISSN
IST-REx-ID
Cite this
Kobayashi W, Michael AK, Ruangroengkulrith S, Kümmecke M, Tachibana K. Protocol for integrative analysis of transcription factor-nucleosome interactions using SeEN-seq and cryo-EM structure determination. STAR Protocols. 2025;7. doi:10.1016/j.xpro.2025.104295
Kobayashi, W., Michael, A. K., Ruangroengkulrith, S., Kümmecke, M., & Tachibana, K. (2025). Protocol for integrative analysis of transcription factor-nucleosome interactions using SeEN-seq and cryo-EM structure determination. STAR Protocols. Elsevier. https://doi.org/10.1016/j.xpro.2025.104295
Kobayashi, Wataru, Alicia K. Michael, Siwat Ruangroengkulrith, Maximilian Kümmecke, and Kikuë Tachibana. “Protocol for Integrative Analysis of Transcription Factor-Nucleosome Interactions Using SeEN-Seq and Cryo-EM Structure Determination.” STAR Protocols. Elsevier, 2025. https://doi.org/10.1016/j.xpro.2025.104295.
W. Kobayashi, A. K. Michael, S. Ruangroengkulrith, M. Kümmecke, and K. Tachibana, “Protocol for integrative analysis of transcription factor-nucleosome interactions using SeEN-seq and cryo-EM structure determination,” STAR Protocols, vol. 7. Elsevier, 2025.
Kobayashi W, Michael AK, Ruangroengkulrith S, Kümmecke M, Tachibana K. 2025. Protocol for integrative analysis of transcription factor-nucleosome interactions using SeEN-seq and cryo-EM structure determination. STAR Protocols. 7, 104295.
Kobayashi, Wataru, et al. “Protocol for Integrative Analysis of Transcription Factor-Nucleosome Interactions Using SeEN-Seq and Cryo-EM Structure Determination.” STAR Protocols, vol. 7, 104295, Elsevier, 2025, doi:10.1016/j.xpro.2025.104295.
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