The CUT&RUN suspect list of problematic regions of the genome

BackgroundCleavage Under Targets and Release Using Nuclease (CUT & RUN) is an increasingly popular technique to map genome-wide binding profiles of histone modifications, transcription factors, and co-factors. The ENCODE project and others have compiled blacklists for ChIP-seq which have been widely adopted: these lists contain regions of high and unstructured signal, regardless of cell type or protein target, indicating that these are false positives. While CUT & RUN obtains similar results to ChIP-seq, its biochemistry and subsequent data analyses are different. We found that this results in a CUT & RUN-specific set of undesired high-signal regions.ResultsWe compile suspect lists based on CUT & RUN data for the human and mouse genomes, identifying regions consistently called as peaks in negative controls. Using published CUT & RUN data from our and other labs, we show that the CUT & RUN suspect regions can persist even when peak calling is performed with SEACR or MACS2 against a negative control and after ENCODE blacklist removal. Moreover, we experimentally validate the CUT & RUN suspect lists by performing reiterative negative control experiments in which no specific protein is targeted, showing that they capture more than 80% of the peaks identified.ConclusionsWe propose that removing these problematic regions can substantially improve peak calling in CUT & RUN experiments, resulting in more reliable datasets.