[Effect of sonication on results of ChIP-seq experiments involving PRC2 related proteins]

Sonication is one of the essential strategies of chromatin fragmentation in Chromatin immunoprecipitation (ChIP) assay. The impact of proteins with different molecular weights generated under different duration of sonication on the results of Chromatin immunoprecipitation sequencing (ChIP-seq) experiments involving the Polycomb Repressive Complex 2 (PRC2) related protein EZH2, which is a histone methyltransferase, and its product H3K27me3 was investigated. The results indicate that in the promoter region or nonpromoter region, there were hardly any differences among the H3K27me3 peaks annotated genes from different duration of sonication, which suggesting that the duration of sonication had little effect on histone ChIP-seq results. In contrast, in the promoter region newly gained EZH2 peaks annonated genes at 20 min sonication time were significantly clustered in the gene ontology (GO) pathways related to actin filament bundle compared with 10 min. In the nonpromoter region, compared with 10 min, the GO pathways of newly gained EZH2 peaks annonated genes at 20 min sonication time is much more than that of lost EZH2 peaks annonated genes. And in the nonpromoter region, compared with 20 min, the GO pathways of lost EZH2 peaks annonated genes at 30 min sonication time is much more than that of newly gained EZH2 peaks annonated genes. Most of these pathways are associated with RNA polymerase II (RNAPII), organ development and cell morphogenesis. These suggest that the genomic information of EZH2 will be lost if the duration of sonication is not enough or too long. Different duration of sonication mainly affect the EZH2 peaks in PRC2 unoccupied region and the bivalent promoter in the promoter region, as well as the PRC2 occupied region, PRC2 unoccupied region and the activated enhancer in the nonpromoter region. Therefore, the sonication for the chromatin related proteins with high molecular weights need to be optimized to make chromatin fragments size vary from 100 bp to 500 bp, which will yield relatively comprehensive genomic information of the target protein. For histones, which are of small molecular weights, duration of sonication has little effect on them.