Protocol for analysis of G2/M DNA synthesis in human cells.
Protocol for analysis of G2/M DNA synthesis in human cells.
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DOI：
10.1016/j.xpro.2021.100570
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发表时间：
2021-06-18
期刊：
影响因子：
--
通讯作者：
Saponaro M
中科院分区：
文献类型：
--
作者：
Wang J;Saponaro M
在免疫荧光显微镜下，每5个G2/M细胞中就有1个在未受干扰的情况下观察到G2/M DNA合成(G-mids)。然而，对发生G-MID的基因组位置知之甚少。在这里，我们描述了一种允许丰富G2/M细胞并使用BrdU-seq调查G-mid位点的方案。该方法还可用于研究DNA复制或转录相关因子在不同细胞系中影响G-mids水平的作用。有关本议定书的使用和执行的完整细节，请参阅。如何将细胞阻滞在G0/G1期，并同步释放到S期，流式细胞仪鉴定G2/M期细胞，进行DNA合成下拉G2/M期合成的DNA下一代测序G2/MDNA合成位点(G-MIDs)可以在免疫荧光显微镜下观察到五分之一的G2/M细胞在未受干扰的情况下。然而，对发生G-MID的基因组位置知之甚少。在这里，我们描述了一种允许丰富G2/M细胞并使用BrdU-seq调查G-mid位点的方案。该方法还可用于研究DNA复制或转录相关因子在不同细胞系中影响G-mids水平的作用。
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. How to arrest cells in G0/G1 and to get a synchronous release into S-phase FACS strategy to identify cells in G2/M performing DNA synthesis Pull-down of DNA synthesized in G2/M Next-generation sequencing of sites of G2/M DNA synthesis 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.
影响因子：
8.8
作者：
Wang J;Rojas P;Mao J;Mustè Sadurnì M;Garnier O;Xiao S;Higgs MR;Garcia P;Saponaro M
通讯作者：
Saponaro M
影响因子：
14.9
作者：
Afgan E;Baker D;van den Beek M;Blankenberg D;Bouvier D;Čech M;Chilton J;Clements D;Coraor N;Eberhard C;Grüning B;Guerler A;Hillman-Jackson J;Von Kuster G;Rasche E;Soranzo N;Turaga N;Taylor J;Nekrutenko A;Goecks J
通讯作者：
Goecks J
影响因子：
16
作者：
Bhowmick, Rahul;Minocherhomji, Sheroy;Hickson, Ian D.
通讯作者：
Hickson, Ian D.