Targeted in situ genome-wide profiling with high efficiency for low cell numbers

Skene, Peter J.; Henikoff, Steven; Henikoff, Steven

doi:10.1038/nprot.2018.015

Cited by 643 publications

(713 citation statements)

References 51 publications

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“…Similarly, cleavage under targets and release using nuclease (CUT&RUN) is a novel technique for the detection of genome‐wide occupancy in situ . It employs antibody‐targeted micrococcal nuclease to release small DNA fragments that are subject to high‐throughput sequencing.…”

Section: How Does One Detect Binding At Targets Where the Tf Operatesmentioning

confidence: 99%

“…Similarly, cleavage under targets and release using nuclease (CUT&RUN) is a novel technique for the detection of genomewide occupancy in situ. [33,34] It employs antibody-targeted micrococcal nuclease to release small DNA fragments that are subject to high-throughput sequencing. This method requires fewer cells (single-cell CUT&RUN has now also been performed [35] ) and exhibits a better signal-to-noise ratio, as well as better resolution than ChIP-seq.…”

Section: How Does One Detect Binding At Targets Where the Tf Operatesmentioning

confidence: 99%

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Hit and Run Transcriptional Repressors Are Difficult to Catch in the Act

et al. 2019

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Transcriptional silencing may not necessarily depend on the continuous residence of a sequence‐specific repressor at a control element and may act via a “hit and run” mechanism. Due to limitations in assays that detect transcription factor (TF) binding, such as chromatin immunoprecipitation followed by high‐throughput sequencing (ChIP‐seq), this phenomenon may be challenging to detect and therefore its prevalence may be underappreciated. To explore this possibility, erythroid gene promoters that are regulated directly by GATA1 in an inducible system are analyzed. It is found that many regulated genes are bound immediately after induction of GATA1 but the residency of GATA1 decreases over time, particularly at repressed genes. Furthermore, it is shown that the repressive mark H3K27me3 is seldom associated with bound repressors, whereas, in contrast, the active (H3K4me3) histone mark is overwhelmingly associated with TF binding. It is hypothesized that during cellular differentiation and development, certain genes are silenced by repressive TFs that subsequently vacate the region. Catching such repressor TFs in the act of silencing via assays such as ChIP‐seq is thus a temporally challenging prospect. The use of inducible systems, epitope tags, and alternative techniques may provide opportunities for detecting elusive “hit and run” transcriptional silencing. Also see the video abstract here https://youtu.be/vgrsoP_HF3g.

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Section: How Does One Detect Binding At Targets Where the Tf Operatesmentioning

confidence: 99%

Section: How Does One Detect Binding At Targets Where the Tf Operatesmentioning

confidence: 99%

Hit and Run Transcriptional Repressors Are Difficult to Catch in the Act

et al. 2019

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“…Much as in ChIP, chromatin‐bound proteins are identified using a primary antibody directed against the protein of interest. However, CUT&RUN does not require crosslinking or sonication (although crosslinking can be used; Skene, Henikoff, & Henikoff, ). Rather, CUT&RUN utilizes a recombinant protein A–microccocal nuclease (pA‐MN) fusion to specifically digest DNA fragments surrounding the protein of interest.…”

Section: Introductionmentioning

confidence: 99%

High‐Resolution Chromatin Profiling Using CUT&RUN

Hainer

Fazzio

2019

CP Molecular Biology

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Determining the genomic location of DNA‐binding proteins is essential to understanding their function. Cleavage Under Targets and Release Using Nuclease (CUT&RUN) is a powerful method for mapping protein‐DNA interactions at high resolution. In CUT&RUN, a recombinant protein A–microccocal nuclease (pA‐MN) fusion is recruited by an antibody targeting the chromatin protein of interest; this can be done with either uncrosslinked or formaldehyde‐crosslinked cells. DNA fragments near sites of antibody binding are released from the insoluble bulk chromatin through endonucleolytic cleavage and used to build barcoded DNA‐sequencing libraries that can be sequenced in pools of at least 30. Therefore, CUT&RUN provides an alternative to ChIP‐seq approaches for mapping chromatin proteins, which typically have relatively high signal‐to‐noise ratios, while using fewer cells and at a lower cost. Here, we describe the methods for performing CUT&RUN, generating DNA‐sequencing libraries, and analyzing the resulting datasets. © 2019 by John Wiley & Sons, Inc.

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“…MapR was developed as an alternative, antibody-independent method to profile R-loops genome wide and, as such, takes advantage of the natural specificity of RNase H for recognizing DNA:RNA hybrids. MapR is heavily based on CUT&RUN, a new and fast method to identify transcription-factor binding sites genome wide (Skene & Henikoff, 2017;Skene, Henikoff, & Henikoff, 2018).…”

Section: Background Informationmentioning

confidence: 99%

MapR: A Method for Identifying Native R‐Loops Genome Wide

Yan

Sarma

2020

CP Molecular Biology

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R‐loops are abundant, RNA‐containing chromatin structures that form in the genomes of both eukaryotes and prokaryotes. Devising methods to identify the precise genomic locations of R‐loops is critical to understand how these structures regulate numerous cellular processes, including replication, termination, and chromosome segregation, and how their unscheduled formation results in disease. Here, we describe a new, highly sensitive, and antibody‐independent method, MapR, to profile native R‐loops genome wide. MapR takes advantage of the natural specificity of the RNase H enzyme to recognize DNA:RNA hybrids, a defining feature of R‐loops, and combines it with a CUT&RUN approach to target, cleave, and release R‐loops that can then be sequenced. MapR has low background, is faster than current R‐loop detection technologies, and can be performed in any cell type without the need to generate stable cell lines. © 2020 by John Wiley & Sons, Inc.

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Targeted in situ genome-wide profiling with high efficiency for low cell numbers

Cited by 643 publications

References 51 publications

Hit and Run Transcriptional Repressors Are Difficult to Catch in the Act

Hit and Run Transcriptional Repressors Are Difficult to Catch in the Act

High‐Resolution Chromatin Profiling Using CUT&RUN

MapR: A Method for Identifying Native R‐Loops Genome Wide

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