Cancer Research UK, Cambridge Institute, Li Ka Shing Centre, Cambridge, UK.
Department of Chemistry, University of Cambridge, Cambridge, UK.
Nat Protoc. 2018 Mar;13(3):551-564. doi: 10.1038/nprot.2017.150. Epub 2018 Feb 22.
G-rich DNA sequences can form four-stranded G-quadruplex (G4) secondary structures and are linked to fundamental biological processes such as transcription, replication and telomere maintenance. G4s are also implicated in promoting genome instability, cancer and other diseases. Here, we describe a detailed G4 ChIP-seq method that robustly enables the determination of G4 structure formation genome-wide in chromatin. This protocol adapts traditional ChIP-seq for the detection of DNA secondary structures through the use of a G4-structure-specific single-chain antibody with refinements in chromatin immunoprecipitation followed by high-throughput sequencing. This technology does not require expression of the G4 antibody in situ, enabling broad applicability to theoretically all chromatin sources. Beginning with chromatin isolation and antibody preparation, the entire protocol can be completed in <1 week, including basic computational analysis.
富含 G 的 DNA 序列可以形成四链体 G-四链螺旋（G4）二级结构，并与转录、复制和端粒维持等基本生物过程相关联。G4 也与促进基因组不稳定性、癌症和其他疾病有关。在这里，我们描述了一种详细的 G4 ChIP-seq 方法，该方法能够在染色质中稳健地确定全基因组范围内 G4 结构的形成。该方案通过使用 G4 结构特异性单链抗体来适应传统的 ChIP-seq 检测 DNA 二级结构，该抗体在进行染色质免疫沉淀后经过改进，随后进行高通量测序。该技术不需要在原位表达 G4 抗体，从而能够广泛适用于理论上所有的染色质来源。从染色质分离和抗体制备开始，整个方案可以在<1 周内完成，包括基本的计算分析。
