Protocol of the Double-Click Seq Method to Evaluate the Deposition Bias of Chromatin Proteins

Petra E. van der Wouden, Martijn R. H. Zwinderman, Deng Chen, Michela Borghesan, Frank J. Dekker*

*Bijbehorende auteur voor dit werk

OnderzoeksoutputAcademicpeer review

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Samenvatting

Symmetrical deposition of parental and newly synthesized chromatin proteins over both sister chromatids is important for the maintenance of epigenetic integrity. However, the mechanisms to maintain equal distribution of parental and newly synthesized chromatid proteins over sister chromatids remains largely unknown. Here, we describe the protocol for the recently developed double-click seq method that enables mapping of asymmetry in the deposition of parental and newly synthesized chromatin proteins on both sister chromatids in DNA replication. The method involved metabolic labeling of new chromatin proteins with l-Azidohomoalanine (AHA) and newly synthesized DNA with Ethynyl-2′-deoxyuridine (EdU) followed by two subsequent click reactions for biotinylation and subsequently by corresponding separation steps. This enables isolation of parental DNA that was bound to nucleosomes containing new chromatin proteins. Sequencing of these DNA samples and mapping around origins of replication in the cellular DNA enables estimation of the asymmetry in deposition of chromatin proteins over the leading and lagging strand in DNA replication. Altogether, this method contributes to the toolbox to understand histone deposition in DNA replication.

Originele taal-2English
Artikelnummere805
Aantal pagina's17
TijdschriftCurrent Protocols
Volume3
Nummer van het tijdschrift6
DOI's
StatusPublished - jun.-2023

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