Division of Chemical Biology and Medicinal Chemistry, Center for Integrative Chemical Biology and Drug Discovery, Eshelman School of Pharmacy , The University of North Carolina , Chapel Hill , North Carolina 27599 , United States.
Curriculum in Genetics and Molecular Biology , The University of North Carolina , Chapel Hill , North Carolina 27514 , United States.
Biochemistry. 2018 May 15;57(19):2756-2761. doi: 10.1021/acs.biochem.8b00075. Epub 2018 Apr 25.
One of the most sensitive, time-consuming, and variable steps of chromatin immunoprecipitation (ChIP) is chromatin sonication. Traditionally, this process can take hours to properly sonicate enough chromatin for multiple ChIP assays. Further, the length of sheared DNA is often inconsistent. In order to faithfully measure chemical and structural changes at the chromatin level, sonication needs to be reliable. Thus, chromatin fragmentation by sonication represents a significant bottleneck to downstream quantitative analysis. To improve the consistency and efficiency of chromatin sonication, we developed and tested a cavitation enhancing reagent based on sonically active nanodroplets. Here, we show that nanodroplets increase sonication efficiency by 16-fold and provide more consistent levels of chromatin fragmentation. Using the previously characterized chromatin in vivo assay (CiA) platform, we generated two distinct chromatin states in order to test nanodroplet-assisted sonication sensitivity in measuring post-translational chromatin marks. By comparing euchromatin to chemically induced heterochromatin at the same CiA:Oct4 locus, we quantitatively measure the capability of our new sonication technique to resolve differences in chromatin structure. We confirm that nanodroplet-assisted sonication results are indistinguishable from those of samples processed with traditional sonication in downstream applications. While the processing time for each sample was reduced from 38.4 to 2.3 min, DNA fragment distribution sizes were significantly more consistent with a coefficient of variation 2.7 times lower for samples sonicated in the presence of nanodroplets. In conclusion, sonication utilizing the nanodroplet cavitation enhancement reagent drastically reduces the amount of processing time and provides consistently fragmented chromatin of high quality for downstream applications.
在染色质免疫沉淀（ChIP）中，最敏感、最耗时且变化最大的步骤之一是染色质超声处理。传统上，这个过程需要数小时才能适当地超声处理足够的染色质用于多个 ChIP 分析。此外，剪切 DNA 的长度通常不一致。为了忠实地测量染色质水平的化学和结构变化，超声处理需要可靠。因此，染色质的超声破碎是下游定量分析的一个重要瓶颈。为了提高染色质超声处理的一致性和效率，我们开发并测试了一种基于声活性纳米液滴的空化增强试剂。在这里，我们表明纳米液滴可将超声处理效率提高 16 倍，并提供更一致的染色质片段水平。使用先前表征的染色质体内分析（CiA）平台，我们生成了两种不同的染色质状态，以测试纳米液滴辅助超声处理在测量翻译后染色质标记物方面的敏感性。通过比较同一 CiA:Oct4 基因座上的常染色质和化学诱导的异染色质，我们定量测量了我们新的超声处理技术在测量染色质结构差异方面的能力。我们证实，纳米液滴辅助超声处理的结果与传统超声处理的样品在下游应用中的结果无法区分。虽然每个样品的处理时间从 38.4 分钟缩短到 2.3 分钟，但在纳米液滴存在下超声处理的样品的 DNA 片段分布大小显著更加一致，变异系数降低了 2.7 倍。总之，利用纳米液滴空化增强试剂进行超声处理可大大减少处理时间，并为下游应用提供高质量的一致碎片化染色质。
