Chromatin shearing in suspension cell line: A guide for optimization

Abstract

Background Chromatin immunoprecipitation (ChIP) assess DNA-proteins interactions and hence helps to generate intricate relationships and vital information. ChIP is integrated with next-generation sequencing (ChIP-seq) to determine the genomic location of specific proteins or post-translational modifications at an individual locus or genome-wide. Although improved sample preparation and library preparation are present, ChIP-seq still endures the complexity of the protocol. The most sensitive and critical step involves the chromatin fragmentation step. The protocol for chromatin shearing varies with cell type and it is time-consuming, hence protocol standardization is required. Methods and Results In our work, we optimized the parameters required for chromatin shearing in suspension cell (Kasumi-1) using S220 Focused-ultra-sonicator (Covaris). To address this, the protocol starts with the fixation of cells with formaldehyde, cell lysis, and nuclei isolation. Chromatin shearing using various sonication buffers and sonicator parameters was performed to determine the efficient sonication condition. We have found success for sonication at the following settings: PIP of 150 W, DF 7.0%, CPB 200, and water fill level 12 generating fragments of approximately 250–600 bp in 7 min. We determined percentage of SDS (0.15%) and DOC (0.05%) in the sonication buffer was an important variable to achieve the desired fragmentation pattern. Conclusions From our study, we found the optimal percentage of detergents in the sonication buffer and the duration of sonication for resulting desired fragmentation pattern. The fragmentation is critical for good coverage and resolution of data, without losing material due to over-fragmentation, hence shearing determines the success of the experiment.