biostudies-literatureAbbasova LMedical Research CouncilAlzheimer's Association2993https://www.ebi.ac.uk/biostudies/studies/S-EPMC11950320biostudies-literatureUnknown16(1)DNA-protein interactions have traditionally been profiled via chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq). Cleavage Under Targets & Tagmentation (CUT&Tag) is a rapidly expanding technique that enables the profiling of such interactions in situ at high sensitivity. However, thorough evaluation and benchmarking against established ChIP-seq datasets are lacking. Here, we comprehensively benchmarked CUT&Tag for H3K27ac and H3K27me3 against published ChIP-seq profiles from ENCODE in K562 cells. Combining multiple new and published CUT&Tag datasets, there was an average recall of 54% known ENCODE peaks for both histone modifications. We tested peak callers MACS2 and SEACR and identified optimal peak calling parameters. Overall, peaks identified by CUT&Tag reprNature communicationsCUT&Tag recovers up to half of ENCODE ChIP-seq histone acetylation peaks.PMC11950320ADSF-21-829660-CMR/W004984/1Ismail JNHu DSkene NGAbbasova LSchilder BMUrbanaviciute PMarzi SJNott AfalseCUT&Tag recovers up to half of ENCODE ChIP-seq histone acetylation peaks.DNA-protein interactions have traditionally been profiled via chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq). Cleavage Under Targets & Tagmentation (CUT&Tag) is a rapidly expanding technique that enables the profiling of such interactions in situ at high sensitivity. However, thorough evaluation and benchmarking against established ChIP-seq datasets are lacking. Here, we comprehensively benchmarked CUT&Tag for H3K27ac and H3K27me3 against published ChIP-seq profiles from ENCODE in K562 cells. Combining multiple new and published CUT&Tag datasets, there was an average recall of 54% known ENCODE peaks for both histone modifications. We tested peak callers MACS2 and SEACR and identified optimal peak calling parameters. Overall, peaks identified by CUT&Tag repr2025-01-01T00:00:00Z2025 Mar2026-06-02T06:35:07.84Z2026-04-15T03:15:32.414ZS-EPMC119503204014827210.1038/s41467-025-58137-2