Cleavage under target and tagmentation (CUT however, its reproducibility is impacted by a lack of standardization in experimental and analytical procedures. This study identifies four key parameters critical for optimizing CUT default model-based analysis of chromatin immunoprecipitation followed by sequencing (MACS2) scaling causes a paradoxical decrease in peak numbers with decreasing IgG control size, a limitation resolved by the "scale-to-large" option. Third, duplicate removal strategies differentially affect peak callers, with MACS2 performing best using biological reads and sparse enrichment analysis for CUT&RUN (SEACR) relying on technical duplicates for accurate calling. Finally, mild crosslinking with 0.2 mM ethylene glycol bis (succinimidyl succinate) (EGS) for 5 min enhances CTCF detection and reduces variability. Together, these optimizations establish practical guidelines for reliable CUT&Tag experimental design and analysis.
Murray et al. (Mon,) studied this question.