Impact of Chromatin Immunoprecipitation Crosslinking Conditions on Selective Genomic Interactions by NF-?B Family Members
The ability to capture protein: DNA interactions in a quantitative, high-resolution, and temporal manner in the cell context is crucial for elucidating the mechanisms underlying transcriptional regulation.  Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a widely used technique that enables the study of histone modifications and genome-wide interactions of transcription factors and chromatin regulators. Although ChIP-seq generates a vast amount of information, crosslinking conditions need to be carefully evaluated to minimize the capture of non-specific interactions. In this study, we conduct an in-depth analysis to evaluate the impact of crosslinking conditions on ChIP-seq results for two NF-kB family members, RelA and c-Rel. We titrate two commonly used chemical crosslinkers, formaldehyde and DSG. We demonstrate that while raising the crosslinking concentrations increases the total number of binding sites and the strength of the binding signal, high crosslinking conditions lead to an abundance of interactions detected at locations that lack anticipated motifs or annotations to potential NF-kB target genes. Our findings emphasize the importance of a comprehensive evaluation of ChIP-seq results and a cautious evaluation of chemical crosslinking conditions. Moreover, we compare ChIP-seq to CUT&TAG, a newer technique that does not require crosslinking. This comparison revealed strongly overlapping genomic interactions between the two methods. However, both methods display evidence of a large number of non-specific signals that exhibit little overlap between the two methods. Overall design: To investigate the effect of crosslinking on the outcome of ChIP-seq, we performed ChIP-seq for cRel and RelA with varying concentrations of both DSG and PFA.