Abstract 1742: A universal spike-in normalization strategy for CUT&RUN, CUT&Tag and ATAC-seq

The development of simple and efficient genome-wide approaches has recently democratized the study of epigenetics. For instance, CUT&RUN and CUT&Tag were developed as fast and affordable alternatives to ChIP-seq, whereas ATAC-seq readily identifies regions of open chromatin. These methods are ideal to look for genome-wide alterations in chromatin structures but are only semi-quantitative and can miss global changes when performed without normalization spike-ins. In fact, many standard computational pipelines fail to detect a reportedly substantial global H3K27me3 decrease in the presence of GSK126, a therapeutic target for lymphoma and potent inhibitor of the EZH2 methyltransferase. To overcome this challenge, we developed a spike-in approach using Drosophila melanogaster nuclei for normalization of ATAC, CUT&RUN and CUT&Tag experiments using human cells. The addition of the spiked-in nuclei allowed for the detection of the expected global changes in open regions of chromatin (ATAC-seq), H3K27me3 (CUT&Tag), R-loops (CUT&Tag), EED and p53 binding (CUT&RUN) upon treatment of K562 cells with GSK126. By contrast, these same changes were all missed when omitting the spike-in nuclei. This streamlined spike-in normalization strategy can be easily applied to any number of cells and on a large variety of conditions. Citation Format: Sarah Traynor, Bradley Townsley, Julianna Weber, Mary Anne Jelinek, Brian Egan, Benjamin Delatte. A universal spike-in normalization strategy for CUT&RUN, CUT&Tag and ATAC-seq [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1742.