Abstract B004: Analysis of epigenetic marks and mechanisms in disease: Your guide to a successful assay


 Like the chromatin immunoprecipitation (ChIP) assay, Cleavage Under Targets & Release Using Nuclease is a powerful and versatile technique used for probing protein-DNA interactions within the natural chromatin context of the cell. The assay can be combined with downstream qPCR or NG-seq to analyze histone modifications and binding of transcription factors, DNA replication factors, or DNA repair proteins at specific target genes or across the entire genome. It provides a rapid, robust, and true low cell number assay for detection of protein-DNA interactions in the cell. Unlike the ChIP assay, it is free from formaldehyde cross-linking, chromatin fragmentation, and immunoprecipitation. Previously, we have shown that, compared to ChIP, it requires fewer starting cells (100K), has a much faster protocol (one day from cells to DNA), generates lower background signal (requires less sequencing depth), and offers spike-in control DNA for effective normalization of signal between samples and between experiments. We recently updated the CST Assay Kit for use with 5,000-20,000 cells and added protocols for fixed cells and tissue. I will discuss the basics of the assay and important factors to consider when setting up your experiment. In addition, I will provide data showing the versatility of this assay for mapping various histone modifications, transcription factor, and transcription cofactor binding across multiple sample types. Finally, I will discuss how the general protocol is optimized for greater signal to noise ratio, reduced number of starting cells, and provide an alternative digestion method to prepare the input DNA as a critical control of the experiment.
 Citation Format: Christopher C. Comeau, Angela H. Guo, Fang Chen, Christopher J. Fry. Analysis of epigenetic marks and mechanisms in disease: Your guide to a successful assay. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr B004.