Abstract
The successful assembly of nucleosomes following DNA replication is critically important for both the inheritance of epigenetic information and the maintenance of genome integrity. This process, termed DNA replication-coupled (RC) nucleosome assembly, requires that DNA replication and nucleosome assembly function in a highly coordinated fashion to transmit both genetic and epigenetic information. In this chapter, we describe a genome-wide method for measuring nucleosome occupancy patterns on nascent strands, which we have termed Replication-Intermediate Nucleosome Mapping (ReIN-Map), to monitor the RC nucleosome assembly level genome-wide in vivo. This method takes advantage of next-generation sequencing and in vivo labeling of newly synthesized DNA using a thymidine analogue, 5-bromo-2′-deoxyuridine (BrdU), and involves parallel analyses of the nucleosome formation using micrococcal nuclease (MNase) digestion of chromatin (MNase-seq) and of the newly synthesized DNA levels using sonication shearing of chromatin s (Sonication-seq). Replicated chromatin was enriched by immunoprecipitation using antibodies against BrdU (BrdU-IP), which is incorporated into DNA during DNA synthesis; the DNA is then subjected to strand-specific sequencing.
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Acknowledgments
Shaofeng Liu, He Leng, Pu Zheng, and Xuezheng Wang from Li laboratory all contributed to the test of the experimental conditions. We thank Chunhe Yu and Haiyun Gan from Zhiguo Zhang laboratory (Columbia University) for advice on BrdU-IP and the library construction. We thank Kaifu Chen (Institute for Academic Medicine at Houston Methodist and Weill Cornell Medical College) for advice with the bioinformatic analysis. This research was supported by the Grants from National Natural Science Foundation of China (No: 31725015; No. 31830048).
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Xu, Z., Feng, J., Li, Q. (2021). Measuring Genome-Wide Nascent Nucleosome Assembly Using ReIN-Map. In: Xiao, W. (eds) Yeast Protocols. Methods in Molecular Biology, vol 2196. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0868-5_10
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DOI: https://doi.org/10.1007/978-1-0716-0868-5_10
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