Cavitation Enhancement Increases the Efficiency and Consistency of Chromatin Fragmentation from Fixed Cells for Downstream Quantitative Applications.

ABSTRACT

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 CiAOct4 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.