Protocol for analysis of G2/M DNA synthesis in human cells

Jianming Wang; Marco Saponaro

doi:10.1016/j.xpro.2021.100570

Protocol for analysis of G2/M DNA synthesis in human cells

Jianming Wang^*, Marco Saponaro

^*Corresponding author for this work

Cancer and Genomic Sciences

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

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Abstract

G2/M DNA synthesis (G-MiDS) can be observed in one in five G2/M cells in unperturbed conditions by immunofluorescence microscopy. However, little is known of the genomic sites undergoing G-MiDS. Here, we describe a protocol which allows enriching for G2/M cells and investigating the sites of G-MiDS using BrdU-seq. This method can also be used to study the role of DNA replication or transcription-associated factors in affecting G-MiDS levels in different cell lines. For complete details on the use and execution of this protocol, please refer to Wang et al. (2021).

Original language	English
Article number	100570
Journal	STAR Protocols
Volume	2
Issue number	2
Early online date	29 May 2021
DOIs	https://doi.org/10.1016/j.xpro.2021.100570
Publication status	Published - 18 Jun 2021

Bibliographical note

Funding Information:
We thank the Genomics Birmingham for help with NGS library preparation and sequencing samples and Paola Pietroni and Adriana Flores-Langarica from the Flow Cytometry Facility for sorting mitotic cells, all from the University of Birmingham. This work was supported by a University of Birmingham fellowship, the Wellcome Trust ( 202115/Z/16/Z ), the Royal Society ( RG170246 ), and the BBSRC ( BB/S016155/1 ) to M.S.

Keywords

Cell Biology
Cell isolation
Flow Cytometry/Mass Cytometry
Molecular Biology
Sequencing

ASJC Scopus subject areas

General Biochemistry,Genetics and Molecular Biology
General Neuroscience
General Immunology and Microbiology

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10.1016/j.xpro.2021.100570Licence: Creative Commons: Attribution-NonCommercial-NoDerivs (CC BY-NC-ND)

WangJ2021ProtocolFinal published version, 2.62 MBLicence: Creative Commons: Attribution-NonCommercial-NoDerivs (CC BY-NC-ND)

Interplay between RNA Pol II transcription and DNA replication
Saponaro, M. (Principal Investigator)
Biotechnology & Biological Sciences Research Council
1/09/19 → 31/08/23
Project: Research Councils

Cite this

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title = "Protocol for analysis of G2/M DNA synthesis in human cells",

abstract = "G2/M DNA synthesis (G-MiDS) can be observed in one in five G2/M cells in unperturbed conditions by immunofluorescence microscopy. However, little is known of the genomic sites undergoing G-MiDS. Here, we describe a protocol which allows enriching for G2/M cells and investigating the sites of G-MiDS using BrdU-seq. This method can also be used to study the role of DNA replication or transcription-associated factors in affecting G-MiDS levels in different cell lines. For complete details on the use and execution of this protocol, please refer to Wang et al. (2021).",

keywords = "Cell Biology, Cell isolation, Flow Cytometry/Mass Cytometry, Molecular Biology, Sequencing",

author = "Jianming Wang and Marco Saponaro",

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AU - Wang, Jianming

AU - Saponaro, Marco

N1 - Funding Information: We thank the Genomics Birmingham for help with NGS library preparation and sequencing samples and Paola Pietroni and Adriana Flores-Langarica from the Flow Cytometry Facility for sorting mitotic cells, all from the University of Birmingham. This work was supported by a University of Birmingham fellowship, the Wellcome Trust ( 202115/Z/16/Z ), the Royal Society ( RG170246 ), and the BBSRC ( BB/S016155/1 ) to M.S.

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Y1 - 2021/6/18

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AB - G2/M DNA synthesis (G-MiDS) can be observed in one in five G2/M cells in unperturbed conditions by immunofluorescence microscopy. However, little is known of the genomic sites undergoing G-MiDS. Here, we describe a protocol which allows enriching for G2/M cells and investigating the sites of G-MiDS using BrdU-seq. This method can also be used to study the role of DNA replication or transcription-associated factors in affecting G-MiDS levels in different cell lines. For complete details on the use and execution of this protocol, please refer to Wang et al. (2021).

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KW - Flow Cytometry/Mass Cytometry

KW - Molecular Biology

KW - Sequencing

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Protocol for analysis of G2/M DNA synthesis in human cells

Abstract

Bibliographical note

Keywords

ASJC Scopus subject areas

Access to Document

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Projects

Interplay between RNA Pol II transcription and DNA replication

Cite this