Cleavage under target and tagmentation (CUT&Tag) is a widely used method for profiling chromatin occupancy; 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&Tag performance. First, optimal cell number requirements are target and species specific: H3K27me3 detection requires ≥10K cells in both mouse and rat cells, whereas CTCF mapping needs 50K cells in mouse embryonic stem cells and 100K in rat C6 cells, reflecting potential differences in protein abundance and antibody affinity. Second, the peak-calling methodology is crucial; 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.