Benchmark of Chromatin-protein Interaction Methods in Haploid Round Spermatids

Ruolei WangYue WuZe ZhouYicheng MaWeidong ZhangZihang WangWeihan Luo^*Peng Hua^*

• State Key Laboratory of Reproductive Medicine and Offspring Health, Nanjing Medical University, Nanjing, China

Chromatin-protein interactions are fundamental for regulation of gene transcription. While ChIP-seq has long been the gold standard for mapping these interactions, emerging techniques such as CUT&RUN and CUT&Tag, which offer advantages like low-input requirements and high signal-to-noise ratios, have aroused great attention. However, research addressing the potential biases introduced by enzyme-based tagmentation approaches, and comparative assessment with ChIP-seq remain absent. This study aims to systematically evaluate and compare the performance of ChIP-seq, CUT&Tag, and CUT&RUN for profiling genome-wide transcription factors and histone modifications binding. Our analysis revealed that all three methods reliably detect histone modifications and transcription factors enrichment, with CUT&Tag standing out for its comparatively higher signal-to-noise ratio. Detailed peak comparison revealed unique and overlapping enrichment among the three techniques.Additionally, CUT&Tag can identify novel CTCF peaks compared with other two methods. A strong correlation was observed between CUT&Tag signal intensity and chromatin accessibility, highlighting its ability to generate high-resolution signals in accessible regions. The systematic comparison summarizes the differences between CUT&Tag and CUT&RUN in terms of signalto-noise ratio and bias towards accessible chromatin. Considering the experimental procedures, signal specificity, and inherent biases, we recommend tailoring the choice of method to the type of chromatin-protein interaction under study. CUT&Tag offers a promising alternative for applications requiring high sensitivity and reduced background noise.