ChEC-seq2:一种改进的染色体内源裂解测序方法和生物信息学分析管道,用于绘制in vivo蛋白质-DNA相互作用的映射
Jake VanBelzen1, Chengzhe Duan1, Donna Garvey Brickner1
1Department of Molecular Biosciences, Northwestern University.
bioRxiv : the preprint server for biology
|October 31, 2023
概括
我们开发了ChEC-seq2,一种简化的染色体内源性裂变,其次是测序方法,以准确地绘制转录因子结合部位. 这种技术与DoubleChEC管道相结合,增强了DNA结合的特异性,并确定了高可靠性的结合部位.
科学领域:
- 分子生物学分子生物学
- 基因组学就是基因组学.
- 生物化学 生物化学
背景情况:
- 染色体免疫沉 (ChIP) 是识别转录因子 (TF) 结合位点的标准方法,但它缺乏分辨率.
- 高分辨率的方法,如ChIP-exo,染色体内源裂变,其次是测序 (ChEC-seq) 和CUT&RUN,提供了改进的TF占用和结合部位保护洞察力.
- 采用内源性TF与微球菌核酶融合的ChEC-seq,绕过了固定和抗体的需求,但在DNA裂变特异性方面存在担忧.
研究的目的:
- 开发一种简化和更具体的ChEC-seq协议 (ChEC-seq2),用于高分辨率地图化体内DNA结合部位.
- 通过最小化核酶消化和增加分裂DNA产量来增强ChEC-seq的特异性.
- 通过确定酵母中已知的转录因子结合部位来验证ChEC-seq2的准确性.
主要方法:
- 开发ChEC-seq2,一种修改后的ChEC-seq协议,涉及最小化的核酶消化.
- 应用DoubleChEC生物信息管道去除非特定的DNA裂变点.
- 通过分析裂变模式和确定三种酵母转录因子的结合位点来验证ChEC-seq2.
主要成果:
- ChEC-seq2显示了复制物之间的高可重现性和与现有ChEC-seq数据的一致性.
- 双重ChEC管道有效地消除了非特定的分离事件,提高了数据质量.
- ChEC-seq2与DoubleChEC结合,确定了三种酵母TF的高可靠性裂解部位,它们在已知的结合部位和目标基因附近显著丰富.
结论:
- 在体内,ChEC-seq2提供了一种简化且高度特定的方法,用于绘制转录因子结合位点的地图.
- 结合ChEC-seq2和DoubleChEC管道,显著提高了TF绑定站点识别的分辨率和准确性.
- 这种方法为研究转录因子结合特征及其调控作用提供了一个强大的工具.
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