非交联染色质免疫共沉淀及其二代测序技术建库方法的优化

The improvewment of DNA library construction in non-crosslinked chromatin immunoprecipitation coupled with next-generation sequencing

目的优化非交联染色质免疫共沉淀及其二代测序(Native ChIP-seq)技术,简化操作步骤,得到高质量ChIP-seq数据。方法裂解细胞,MNase切割DNA释放核小体,用组蛋白修饰的特异性抗体将组蛋白与DNA的复合物进行免疫共沉淀,蛋白酶K消化后进行DNA纯化,染色质免疫共沉淀(ChIP)产物用Tn5转座酶建库与传统建库两种方法进行文库构建并测序。结果与传统建库方法相比,Tn5转座酶建库经过Tn5片段化后直接进行目的DNA的扩增,操作简单,更加省时,效率更高;IGV可视化信号分布峰图显示两种建库方式获得的富集峰基本相同;两种建库方法获得的测序数据中,Tn5转座酶建库比传统建库获得更多的富集峰;Tn5转座酶建库后结果显示重复性良好,信噪比达到50%以上。结论Tn5转座酶建库能提高建库效率并得到更好的数据质量,适用于组蛋白修饰的检测,为表观遗传研究提供更好的技术选择。

Objective To optimize DNA library construction in non-crosslinked chromatin immunoprecipitation coupled with next-generation sequencing (Native ChIP-seq) to obtain high-quality Native ChIP-seq data.Methods Human nasopharyngeal carcinoma HONE1 cell lysate was digested with MNase for release of the nucleosomes,and the histone-DNA complexes were immunoprecipitated with specific antibodies.The protein component in the precipitate was digested with proteinase K followed by DNA purification;the DNA library was constructed for sequence analysis.Results Compared with the conventional DNA library construction,Tn5 transposase method allowed direct enrichment of the target DNA after Tn5 fragmentation,which was simple,time-saving and more efficient.The IGV visualized map showed that the information obtained by the two library construction methods was consistent.The sequencing data obtained by the two methods revealed more signal enrichment with Tn5 transposase library construction than with the conventional approach.H3K4me3 ChIP results showed a good reproducibility after Tn5 transposase library construction with a signal-to-noise ratio above 50%. Conclusion Tn5 transposase method improves the efficiency of DNA library construction and the results of subsequent sequence analysis,and is especially suitable for detecting histone modification in the DNA to provide a better technical option for epigenetic studies.