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CUT&RUN sequencing
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CUT&RUN sequencing
CUT&RUN sequencing, also known as cleavage under targets and release using nuclease, is a method used to analyze protein interactions with DNA. CUT&RUN sequencing combines antibody-targeted controlled cleavage by micrococcal nuclease with massively parallel DNA sequencing to identify the binding sites of DNA-associated proteins. It can be used to map global DNA binding sites precisely for any protein of interest. Currently, ChIP-Seq is the most common technique utilized to study protein–DNA relations, however, it suffers from a number of practical and economical limitations that CUT&RUN sequencing does not.
CUT&RUN sequencing can be used to examine gene regulation or to analyze transcription factor and other chromatin-associated protein binding. Protein-DNA interactions regulate gene expression and are responsible for many biological processes and disease states. This epigenetic information is complementary to genotype and expression analysis. CUT&RUN is an alternative to the current standard of ChIP-seq. ChIP-Seq suffers from limitations due to the cross linking step in ChIP-Seq protocols that can promote epitope masking and generate false-positive binding sites. As well, ChIP-seq suffers from suboptimal signal-to-noise ratios and poor resolution. CUT&RUN sequencing has the advantage of being a simpler technique with lower costs due to the high signal-to-noise ratio, requiring less depth in sequencing.
Specific DNA sites in direct physical interaction with proteins such as transcription factors can be isolated by Protein-A (pA) conjugated micrococcal nuclease (MNase) bound to a protein of interest. MNase mediated cleavage produces a library of target DNA sites bound to a protein of interest in situ. Sequencing of prepared DNA libraries and comparison to whole-genome sequence databases allows researchers to analyze the interactions between target proteins and DNA, as well as differences in epigenetic chromatin modifications. Therefore, the CUT&RUN method may be applied to proteins and modifications, including transcription factors, polymerases, structural proteins, protein modifications, and DNA modifications.
CUT&RUN is an adaptation and improvement on chromatin endogenous cleavage (ChEC) which uses a DNA-binding protein genetically fused to micrococcal nuclease (MNase). These transcription factor-MNase fusion proteins can cleave DNA around the DNA-binding site of the protein of interest. In the adapted process, purified MNase is tagged with Protein A (pA) which targets an antibody that has been added to the cell and is specific for the DNA-binding protein that is of interest. There are seven general steps to the CUT&RUN process.
The first step required is the hypotonic lysis of the cells of interest to isolate the nuclei. The nuclei are then centrifuged, washed in a buffer solution, complexed with lectin-coated magnetic beads. The Lectin-Nuclei complex is then resuspended with an antibody targeted at the protein of interest. The antibody and nuclei are then incubated in the buffer for approximately 2 hours before the nuclei are washed in buffer to remove unbound antibodies. Next, the nuclei are resuspended in the buffer with Protein-A-MNase and are incubated for 1 hour. The nuclei are then again washed in buffer to remove any unbound protein-A-MNase. Next, the nuclei in tubes are placed in a metal block and placed in ice-water and CaCl2 is added to initiate the calcium dependent nuclease activity of MNase to cleave the DNA around the DNA-binding protein. The protein-A-MNase reaction is quenched by adding chelating agents (EDTA and EGTA). The cleaved DNA fragments are then liberated into the supernatant by incubating the nuclei for an hour before the nuclei is pelleted by centrifugation. The DNA fragments are then extracted from the supernatant and can be used to construct a sequencing library.
Unlike ChIP-Seq there is no size selection required before sequencing. A single sequencing run can scan for genome-wide associations with high resolution, due to the low background achieved by performing the reaction in situ with the CUT&RUN sequencing methodology. ChIP-Seq, by contrast, requires ten times the sequencing depth because of the intrinsically high background associated with the method. The data is then collected and analyzed using software that aligns sample sequences to a known genomic sequence to identify the CUT&RUN DNA fragments.
There are detailed CUT&RUN workflows available in an open-access methods repository.
CUT&RUN sequencing provides low levels of background signal because of in situ profiling which retains in vivo 3D confirmations of transcription factor-DNA interactions, so antibodies access only exposed surfaces. Sensitivity of sequencing depends on the depth of the sequencing run (i.e. the number of mapped sequence tags), the size of the genome and the distribution of the target factor. The sequencing depth is directly correlated with cost and negatively correlated with background. Therefore, low-background CUT&RUN sequencing is inherently more cost-effective than high-background ChIP-Sequencing.
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CUT&RUN sequencing AI simulator
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CUT&RUN sequencing
CUT&RUN sequencing, also known as cleavage under targets and release using nuclease, is a method used to analyze protein interactions with DNA. CUT&RUN sequencing combines antibody-targeted controlled cleavage by micrococcal nuclease with massively parallel DNA sequencing to identify the binding sites of DNA-associated proteins. It can be used to map global DNA binding sites precisely for any protein of interest. Currently, ChIP-Seq is the most common technique utilized to study protein–DNA relations, however, it suffers from a number of practical and economical limitations that CUT&RUN sequencing does not.
CUT&RUN sequencing can be used to examine gene regulation or to analyze transcription factor and other chromatin-associated protein binding. Protein-DNA interactions regulate gene expression and are responsible for many biological processes and disease states. This epigenetic information is complementary to genotype and expression analysis. CUT&RUN is an alternative to the current standard of ChIP-seq. ChIP-Seq suffers from limitations due to the cross linking step in ChIP-Seq protocols that can promote epitope masking and generate false-positive binding sites. As well, ChIP-seq suffers from suboptimal signal-to-noise ratios and poor resolution. CUT&RUN sequencing has the advantage of being a simpler technique with lower costs due to the high signal-to-noise ratio, requiring less depth in sequencing.
Specific DNA sites in direct physical interaction with proteins such as transcription factors can be isolated by Protein-A (pA) conjugated micrococcal nuclease (MNase) bound to a protein of interest. MNase mediated cleavage produces a library of target DNA sites bound to a protein of interest in situ. Sequencing of prepared DNA libraries and comparison to whole-genome sequence databases allows researchers to analyze the interactions between target proteins and DNA, as well as differences in epigenetic chromatin modifications. Therefore, the CUT&RUN method may be applied to proteins and modifications, including transcription factors, polymerases, structural proteins, protein modifications, and DNA modifications.
CUT&RUN is an adaptation and improvement on chromatin endogenous cleavage (ChEC) which uses a DNA-binding protein genetically fused to micrococcal nuclease (MNase). These transcription factor-MNase fusion proteins can cleave DNA around the DNA-binding site of the protein of interest. In the adapted process, purified MNase is tagged with Protein A (pA) which targets an antibody that has been added to the cell and is specific for the DNA-binding protein that is of interest. There are seven general steps to the CUT&RUN process.
The first step required is the hypotonic lysis of the cells of interest to isolate the nuclei. The nuclei are then centrifuged, washed in a buffer solution, complexed with lectin-coated magnetic beads. The Lectin-Nuclei complex is then resuspended with an antibody targeted at the protein of interest. The antibody and nuclei are then incubated in the buffer for approximately 2 hours before the nuclei are washed in buffer to remove unbound antibodies. Next, the nuclei are resuspended in the buffer with Protein-A-MNase and are incubated for 1 hour. The nuclei are then again washed in buffer to remove any unbound protein-A-MNase. Next, the nuclei in tubes are placed in a metal block and placed in ice-water and CaCl2 is added to initiate the calcium dependent nuclease activity of MNase to cleave the DNA around the DNA-binding protein. The protein-A-MNase reaction is quenched by adding chelating agents (EDTA and EGTA). The cleaved DNA fragments are then liberated into the supernatant by incubating the nuclei for an hour before the nuclei is pelleted by centrifugation. The DNA fragments are then extracted from the supernatant and can be used to construct a sequencing library.
Unlike ChIP-Seq there is no size selection required before sequencing. A single sequencing run can scan for genome-wide associations with high resolution, due to the low background achieved by performing the reaction in situ with the CUT&RUN sequencing methodology. ChIP-Seq, by contrast, requires ten times the sequencing depth because of the intrinsically high background associated with the method. The data is then collected and analyzed using software that aligns sample sequences to a known genomic sequence to identify the CUT&RUN DNA fragments.
There are detailed CUT&RUN workflows available in an open-access methods repository.
CUT&RUN sequencing provides low levels of background signal because of in situ profiling which retains in vivo 3D confirmations of transcription factor-DNA interactions, so antibodies access only exposed surfaces. Sensitivity of sequencing depends on the depth of the sequencing run (i.e. the number of mapped sequence tags), the size of the genome and the distribution of the target factor. The sequencing depth is directly correlated with cost and negatively correlated with background. Therefore, low-background CUT&RUN sequencing is inherently more cost-effective than high-background ChIP-Sequencing.