Analysis of Epigenetic Marks and Mechanisms in Disease:Your Guide to a Successful CUT&RUN Assay.
Fang Chen, Christopher R. Comeau, Jillian Mason, Carrie A. Brown, and Christopher J. Fry
Cell Signaling Technology, Inc., Danvers MA 01923
ABSTRACT

CUT&RUN Method Overview

Like the chromatin immunoprecipitation (ChIP) assay, Cleavage Under Targets &Release Using Nuclease (CUT&RUN) is a powerful and versatile technique used forprobing protein-DNA interactions within the natural chromatin context of the cell.
The CUT&RUN assay can be combined with downstream qPCR or NG-seq toanalyze histone modifications and binding of transcription factors, DNA
replication factors, or DNA repair proteins at specific target genes or across theentire genome.

Antibody and pAG-MNase Binding
1.
Cells are immobilized on
Concanavalin A Magnetic Beads toallow for subsequent buffer andreagent exchanges.
2.
Cell membranes are thenpermeabilized with digitonin tofacilitate the entry of primary antibodyand pAG-MNase fusion enzyme into
the cell nuclei.
3.
The target-specific primary antibody
recruits the pAG-MNase to thechromatin through protein-protein
interactions between the antibody andthe pAG domain of the fusion enzyme.

CUT&RUN provides a rapid, robust, and true low cell number assay for detectionof protein-DNA interactions in the cell. Unlike the ChIP assay, CUT&RUN is freefrom formaldehyde cross-linking, chromatin fragmentation, andimmunoprecipitation, making it a much faster and more efficient method forenriching protein-DNA interactions and identifying target genes. CUT&RUN can be
performed in as little as one to two days, from live cells to purified DNA, and has
been shown to work with as few as 500-1000 cells per assay (1,2). Instead offragmenting all of the cellular chromatin as done in ChIP, CUT&RUN utilizes anantibody-targeted digestion of chromatin, resulting in much lower backgroundsignal than seen in the ChIP assay. As a result, CUT&RUN requires only 1/10th thesequencing depth that is required for ChIP-seq assays. Finally, the inclusion ofsimple spike-in control DNA allows for accurate quantification and normalizationof target-protein binding between samples. This provides for effective
normalization of signal between samples and between experiments.

MNase Digestion:
4. The addition of Ca2+ activates the pAGMNase, which gently cleaves andliberates the desired chromatin
fragments, allowing them to diffuse
away from the genomic chromatin, outof the cell, and into the supernatant.

•FEATURED CST REAGENTS

Low cell number: Only 100K cells used to generate all CUT&RUN data shown
compared to 4M cells used to generate ChIP and ChIP-seq data.

•Get results quickly: 1-2 days from cells to DNA.

•Save on sequencing costs: Only requires 3-5M reads due to low background.

•Target versatility: Generate qPCR or NG-seq data for histones, transcription
factors, and cofactors•

Reproducibility: Spike-In DNA control allows for normalization betweensamples•

Avoid artifacts: in vivo method using native, NOT cross-linked chromatin• DNA Purification Buffers and Spin Columns (ChIP andCUT&RUN) #14209• SimpleChIP® Universal qPCR Master Mix #88989• SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina®
• SimpleChIP® ChIP-seq Mutiplex Oligos for Illumina® (DualIndex Primers)• SimpleChIP® ChIP-seq Multiplex Oligos for Illumina® (SingleIndex Primers)

CUT&RUN and ChIP assays were performed with HCT 116 cells using the CUT&RUN
Assay Kit #86652 (left panel) or SimpleChIP® Plus Enzymatic Chromatin IP Kit(Magnetic Beads) #9005 (right panel). The enriched DNA was quantified by real-time
PCR using human c-Myc promoter primers, SimpleChIP® Human H19/Igf2 ICR Primers#5172, and SimpleChIP® Human α Satellite Repeat Primers #4486. The amount of
immunoprecipitated DNA in each sample is represented as signal relative to the total
amount of input chromatin, which is equivalent to one.

DNA Purification:
5. DNA is purified using DNA purification spincolumns or phenol/chloroform extraction
followed by ethanol precipitation. Thepurified, enriched DNA is then identified
and quantified using qPCR or NG-seq.

I will discuss the basics of the CUT&RUN assay and important factors to consider
when setting up your experiment. In addition, I will introduce Cell Signaling
Technology’s new CUT&RUN Assay Kit and CUT&RUN pAG-MNase enzyme, andpresent data showing the versatility of this assay for mapping various histone
modifications, transcription factor, and transcription cofactor binding acrossmultiple cell types.

• CUT&RUN Assay Kit #86652• CUT&RUN pAG-MNase and Spike-In DNA #40366

CTCF (D31H2) XP® Rabbit mAb #3418

Advantages

Tri-Methyl-Histone H3 (Lys4) (C42D8)
Rabbit mAb #9751

CUT&RUN DNA Purification(Spin Columns vs. Phenol Chloroform)

Comparison of DNA purification using spin columns or phenol/chloroform
extraction followed by ethanol precipitation. (A) A low range DNA ladder mix (lane 1,unpurified) was purified using either DNA Purification Buffers and Spin Columns (ChIPand CUT&RUN) #14209 (lane 2) or phenol/chloroform extraction followed by ethanol
precipitation (lane 3) and separated by electrophoresis on a 4% agarose gel. As shown,phenol/chloroform followed by ethanol precipitation efficiently recovers all DNA fragment
sizes, while DNA spin columns recover DNA fragments ≥35 bp. (B) DNA was purified
using phenol/chloroform extraction followed by ethanol precipitation from a CUT&RUN
assay performed using TCF4/TCF7L2 (C48H11) Rabbit mAb #2569. The size of theDNA fragments in the library was analyzed using a Bioanalyzer (Agilent Technologies).
The adaptor and barcode sequences added to the library during construction account for
140 bp in fragment length. Therefore, starting 35 bp DNA fragments would be 175 bp inlength after library preparation (indicated with blue vertical line in figure). As shown, lessthan 2% of the total CUT&RUN enriched DNA fragments are less than 175 bp (starting
length of 35 bp), suggesting that DNA purification spin columns are sufficient for captureof >98% of the total CUT&RUN DNA fragments.

CUT&RUN Sample Normalization
Using Spike-In DNA

CUT&RUN and ChIP assays were performed with HCT 116 cells using the CUT&RUN
Assay Kit #86652 or SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads)#9005. DNA Libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit
for Illumina® #56795. The upper panel compares enrichment of CTCF acrosschromosome 8, while the lower panel compares enrichment at the MYC gene, a known
target of CTCF. The input tracks are from the CUT&RUN input sample.

Rpb1 CTD (4H8) Mouse mAb #2629DESCRIPTION
The CUT&RUN Assay Kit is designed to conveniently provide reagents needed toperform up to 24 digestion reactions from cells and is optimized for 100,000 cells
per reaction. The kit has been optimized to work for all types of DNA binding
proteins, including histones, transcription factors and cofactors. A complete assay
can be performed in as little as one to two days. This kit is compatible with bothqPCR and Next Generation sequencing (NG-seq).
When performing the CUT&RUN assay, there is no need to fix the cells. Instead,
live cells are permeabilized with digitonin to allow for subsequent diffusion ofprimary antibody and Protein A-Protein G-Micrococcal Nuclease (pAG-MNase)fusion enzyme into the cell nuclei. Once inside the cell nuclei, the target-specificantibody seeks out and binds to the chromatin-bound target protein. The targetbound antibody recruits the pAG-MNase enzyme to the chromatin, where uponactivation of the enzyme with Ca2+ ion, the enzyme gently cleaves and liberates
the desired chromatin fragments, which then diffuse away from the genomic
chromatin, out of the cell, and into the supernatant. These chromatin fragments
are collected from the supernatant and purified using DNA purification spincolumns or phenol/chloroform followed by ethanol precipitation. The enriched
DNA is identified and quantified using real-time quantitative PCR (qPCR) or NGseq.

Normalization of CUT&RUN signals using spike in DNA for qPCR analysis.
CUT&RUN was performed with a decreasing number of HCT 116 using the CUT&RUN
Assay Kit #86652. Enriched DNA was quantified by real-time PCR using SimpleChIP®
Human GAPDH Exon 1 Primers #5516, SimpleChIP® Human β-Actin Promoter Primers#13653, SimpleChIP® Human β-Actin 3’ UTR Primers #13669, and SimpleChIP® Human
MyoD1 Exon 1 Primers #4490. The amount of immunoprecipitated DNA in each sample
is represented as signal relative to the total amount of input chromatin for 100,000 cells.
Non-normalized enrichments are depicted in the left panels. The Sample Normalization
Spike-In DNA was added into each reaction proportionally to the starting cell number.
Based on the difference of qPCR signals from spike in DNA in each sample, CUT&RUNsignals were normalized to the sample containing 100,000 cells. Normalizedenrichments are depicted in the right panels.
CUT&RUN and ChIP assays were performed with HCT 116 cells using the CUT&RUN
Assay Kit #86652 (left panel) or SimpleChIP® Plus Enzymatic Chromatin IP Kit(Magnetic Beads) #9005 (right panel). The enriched DNA was quantified by real-time
PCR using SimpleChIP® Human GAPDH Exon 1 Primers #5516 and SimpleChIP®
Human α Satellite Repeat Primers #4486. The amount of immunoprecipitated DNA ineach sample is represented as signal relative to the total amount of input chromatin,
which is equivalent to one.

CUT&RUN and ChIP assays were performed with HeLa cells using the CUT&RUN
Assay Kit #86652 (left panel) or the SimpleChIP® Plus Enzymatic Chromatin IP Kit(Magnetic Beads) #9005 (right panel). The enriched DNA was quantified by real-time
PCR using SimpleChIP® Human RPL30 Exon 3 Primers #7014, SimpleChIP® HumanGAPDH Exon 1 Primers #5516, and SimpleChIP® Human MyoD1 Exon1 Primers #4490.
The amount of immunoprecipitated DNA in each sample is represented as signal relative
to the total amount of input chromatin, which is equivalent to one.

The CUT&RUN Assay Kit also provides important controls to ensure a successful
CUT&RUN experiment. The kit contains a positive control Tri-Methyl-Histone H3(Lys4) (C42D8) Rabbit mAb #9751 and a negative control Rabbit (DA1E) mAb IgG
XP® Isotype Control (CUT&RUN) #66362, both of which can be used for qPCR or
NG-seq analysis. PCR primer sets are provided for the human (#7014) and mouse(#7015) RPL30 gene locus to be used in conjunction with the control antibodies.

A.

B.

REFERENCES
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4.

Skene, J.S. and Henikoff, S. (2017) eLIFE 6, e21856.
Skene, J.S. et al. (2018) Nature Protocols 13, 1006-1019.
Meers, M.P. et al. (2019) eLIFE 8, e46314.
Meers, M.P. et al. (2019) Molecular Cell 75, 1-14.

CUT&RUN and ChIP assays were performed with HCT 116 cells using the CUT&RUN
Assay Kit #86652 or Simple-ChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads)#9005. DNA Libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kitfor Illumina® #56795. The upper panel compares enrichment of H3K4me3 acrosschromosome 12, while the lower panel compares enrichment at the GAPDH gene, aknown target of H3K4me3. The input tracks are from the CUT&RUN input sample.

CUT&RUN and ChIP assays were performed with HeLa cells using the CUT&RUN
Assay Kit #86652 or the SimpleChIP® Plus Enzymatic Chromatin IP Kit (MagneticBeads) #9005. DNA Libraries were prepared using SimpleChIP® ChIP-seq DNA Library
Prep Kit for Illumina® #56795. The upper panel compares enrichment of Rpb1 acrosschromosome 12, while the lower panel compares enrichment at the GAPDH gene, aknown target of Rbp1. The input tracks are from the CUT&RUN input sample.

For Research Use Only. Not For Use in Diagnostic Procedures©2020 Cell SignalingTechnology, Inc. Cell Signaling Technology, CST, and SimpleChIP are trademarks of Cell Signaling Technology, Inc.
©2020 Illumina and NextSeq are trademarks of Illumina, Inc.

CUT&RUN assays were performed with NCCIT cells using the CUT&RUN Assay Kit#86652. DNA Libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep
Kit for Illumina® #56795. NG-seq tracks are shown for Ezh2 (D2C9) XP® Rabbit mAb#5246 and SUZ12 (D39F6) XP® Rabbit mAb #3737 (A) or SMARCC1/BAF155 (D7F8S)
Rabbit mAb #11956 and Brg1 (D1Q7F) Rabbit mAb #49360 (B).

Christopher J Fry
email: cfry@cellsignal.com
www.cellsignal.com/posters