MilliporeSigma

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

Genome biology (2023-08-11)
Anna Nordin, Gianluca Zambanini, Pierfrancesco Pagella, Claudio Cantù
ABSTRACT

Cleavage 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. We 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. We propose that removing these problematic regions can substantially improve peak calling in CUT&RUN experiments, resulting in more reliable datasets.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
CHIR99021, ≥98% (HPLC)
Sigma-Aldrich
Anti-HA Tag Antibody, clone 114-2C-7, rabbit monoclonal, clone 114-2C-7, Upstate®, from rabbit