Chromatin Shearing in Suspension Cell Line: A Guide for Optimization.

Abstract

Chromatin immunoprecipitation (ChIP) assesses DNA-proteins interactions and hence helps to generate intricate relationships and vital information. ChIP determines the genomic location of specific proteins or post-translational modifications at an individual locus or genome-wide. The protocol endures complexity; hence it is of utmost importance to identify the variable responsible for experimental erraticism. The most sensitive and critical step involves the chromatin fragmentation step. In the current study, the parameters required for chromatin shearing in the Kasumi-1 cell line have been optimized. To address this, the protocol includes the fixation of cells with formaldehyde followed by cell lysis and nuclei isolation. Further chromatin shearing using various sonication buffers and sonicator parameters was performed. Successful sonication was observed at the following settings: peak incident power of 150 W, duty factor 7.0%, cycles per burst 200, and water fill level 8 generating fragments of ∼250-600 bp in 7 min. To analyze enriched DNA sequences that are associated with the target protein ChIP coupled with quantitative PCR was performed. With this study, the optimal procedure has been standardized for a percentage of detergents, SDS (0.15%), DOC (0.05%) in the sonication buffer, and duration of sonication to achieve the desired fragmentation pattern. The quality of shearing determines the success of the experiment.