Dataset Information

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

ABSTRACT:

Background

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.

Results

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.

Conclusions

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

SUBMITTER: Nordin A

PROVIDER: S-EPMC10416431 | biostudies-literature | 2023 Aug

REPOSITORIES: biostudies-literature

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Publications

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

Nordin Anna A Zambanini Gianluca G Pagella Pierfrancesco P Cantù Claudio C

Genome biology 20230810 1

<h4>Background</h4>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 result ...[more]

PMID: 37563719

Similar Datasets

Project description:Cleavage under targets and release using nuclease (CUT&RUN) is a recently developed chromatin profiling technique that uses a targeted micrococcal nuclease cleavage strategy to obtain high-resolution binding profiles of protein factors or to map histones with specific post-translational modifications. Due to its high sensitivity, CUT&RUN allows quality binding profiles to be obtained with only a fraction of the starting material and sequencing depth typically required for other chromatin profiling techniques such as chromatin immunoprecipitation. Although CUT&RUN has been widely adopted in multiple model systems, it has rarely been utilized in Caenorhabditis elegans, a model system of great importance to genomic research. Cell dissociation techniques, which are required for this approach, can be challenging in C. elegans due to the toughness of the worm's cuticle and the sensitivity of the cells themselves. Here, we describe a robust CUT&RUN protocol for use in C. elegans to determine the genome-wide localization of protein factors and specific histone marks. With a simple protocol utilizing live, uncrosslinked tissue as the starting material, performing CUT&RUN in worms has the potential to produce physiologically relevant data at a higher resolution than chromatin immunoprecipitation. This protocol involves a simple dissociation step to uniformly permeabilize worms while avoiding sample loss or cell damage, resulting in high-quality CUT&RUN profiles with as few as 100 worms and detectable signal with as few as 10 worms. This represents a significant advancement over chromatin immunoprecipitation, which typically uses thousands or hundreds of thousands of worms for a single experiment. The protocols presented here provide a detailed description of worm growth, sample preparation, CUT&RUN workflow, library preparation for high-throughput sequencing, and a basic overview of data analysis, making CUT&RUN simple and accessible for any worm lab. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Growth and synchronization of C. elegans Basic Protocol 2: Worm dissociation, sample preparation, and optimization Basic Protocol 3: CUT&RUN chromatin profiling Alternate Protocol: Improving CUT&RUN signal using a secondary antibody Basic Protocol 4: CUT&RUN library preparation for Illumina high-throughput sequencing Basic Protocol 5: Basic data analysis using Linux.

Dataset Information

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

Background

Results

Conclusions

Publications

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

Similar Datasets

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