Protocol: A Multiplexed Reporter Assay to Study Effects of Chromatin Context on DNA Double-Strand Break Repair

Ruben Schep; Christ Leemans; Eva K Brinkman; Tom van Schaik; Bas van Steensel

doi:10.3389/fgene.2021.785947

Protocol: A Multiplexed Reporter Assay to Study Effects of Chromatin Context on DNA Double-Strand Break Repair

Ruben Schep, Christ Leemans, Eva K Brinkman, Tom van Schaik, Bas van Steensel^*

^*Corresponding author for this work

Cell biology

Research output: Contribution to journal › Article › Academic › peer-review

4 Citations (Scopus)

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Abstract

DNA double-strand breaks (DSBs) can be repaired through various pathways. Understanding how these pathways are regulated is of great interest for cancer research and optimization of gene editing. The local chromatin environment can affect the balance between repair pathways, but this is still poorly understood. Here we provide a detailed protocol for DSB-TRIP, a technique that utilizes the specific DNA scars left by DSB repair pathways to study pathway usage throughout the genome. DSB-TRIP randomly integrates a repair reporter into many genomic locations, followed by the induction of DSBs in the reporter. Multiplexed sequencing of the resulting scars at all integration sites then reveals the balance between several repair pathways, which can be linked to the local chromatin state of the integration sites. Here we present a step-by-step protocol to perform DSB-TRIP in K562 cells and to analyse the data by a dedicated computational pipeline. We discuss strengths and limitations of the technique, as well as potential additional applications to study DNA repair.

Original language	English
Article number	785947
Journal	Frontiers in Genetics
Volume	12
DOIs	https://doi.org/10.3389/fgene.2021.785947
Publication status	Published - 31 Jan 2022

Bibliographical note

Funding Information:
Supported by ZonMW-TOP grant 91215067, ERC Advanced Grant 694466, and NIH Common Fund “4D Nucleome” Program grant U54DK107965. The Oncode Institute is partly supported by KWF Dutch Cancer Society.

Funding Information:
Supported by ZonMW-TOP grant 91215067, ERC Advanced Grant 694466, and NIH Common Fund “4D Nucleome”

Publisher Copyright:
Copyright © 2022 Schep, Leemans, Brinkman, van Schaik and van Steensel.

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10.3389/fgene.2021.785947Licence: CC BY

Protocol A Multiplexed Reporter Assay to Study Effects of Chromatin Context on DNA Double-Strand Break RepairFinal published version, 1.43 MBLicence: CC BY

Cite this

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abstract = "DNA double-strand breaks (DSBs) can be repaired through various pathways. Understanding how these pathways are regulated is of great interest for cancer research and optimization of gene editing. The local chromatin environment can affect the balance between repair pathways, but this is still poorly understood. Here we provide a detailed protocol for DSB-TRIP, a technique that utilizes the specific DNA scars left by DSB repair pathways to study pathway usage throughout the genome. DSB-TRIP randomly integrates a repair reporter into many genomic locations, followed by the induction of DSBs in the reporter. Multiplexed sequencing of the resulting scars at all integration sites then reveals the balance between several repair pathways, which can be linked to the local chromatin state of the integration sites. Here we present a step-by-step protocol to perform DSB-TRIP in K562 cells and to analyse the data by a dedicated computational pipeline. We discuss strengths and limitations of the technique, as well as potential additional applications to study DNA repair.",

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Protocol: A Multiplexed Reporter Assay to Study Effects of Chromatin Context on DNA Double-Strand Break Repair

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