CUT&Tag: An Introduction

 CUT&RUN, Cleavage Under Targets & Tagmentation (CUT&Tag) is a faster, more cost-friendly alternative to ChIP-seq. Compared to ChIP-Seq, CUT&Tag profiles chromatin with less sample, a simplified workflow, lower sequencing costs, and improved signal-to-noise due to lower backgrounds. CUT&Tag is faster than CUT&RUN because most of the library prep occurs in vivo, delivers equivalent data to CUT&RUN, and enables single-cell CUT&Tag.

What Makes CUT&Tag Different

CUT&Tag and CUT&RUN can each be used to profile native chromatin, whereas ChIP and ChIP-seq traditionally utilize cross-linked chromatin. For both CUT&Tag and CUT&RUN, the cellular membrane is permeabilized so the primary antibody can enter the nucleus through the nuclear pore, where they bind to the target of interest. The appropriate pAG-enzyme binds to the antibody and, when activated, cuts the DNA on either side of where the antibody is bound.

Figure 1. The CUT&Tag workflow (left) delivers robust protein-DNA interaction data while decreasing DNA library prep time.

How does CUT&Tag differ from CUT&RUN?

• CUT&Tag utilizes a secondary incubation step after the primary antibody incubation step to boost signal strength.
• CUT&Tag is performed under high salt conditions. Both methods are compatible with histone analysis. However, CUT&Tag is less compatible with transcription factors and cofactors because high salt conditions can interfere with target protein-DNA interactions. This is particularly true for less abundant or weakly bound targets.
• CUT&RUN uses Ca²⁺-activated pAG-MNase to cleave the DNA while CUT&Tag uses Mg²⁺-activated pAG-Tn5 to cleave the DNA. The Tn5 is charged with Illumina adaptors that are added to the chromatin during the cleavage process.
• CUT&Tag permeabilizes both the cellular and nuclear membrane after tagmentation to completely solubilize the CUT&Tag DNA. This leads to the presence of both tagmented DNA and genomic DNA in the final DNA sample, making CUT&Tag DNA incompatible with qPCR. If qPCR is desired, we recommend performing CUT&RUN.
• CUT&Tag skips the in vitro adaptor ligation step required for CUT&RUN, allowing you to skip directly to PCR amplification of your DNA library for sequencing–saving you precious time.
• CUT&Tag’s time savings is cumulative, meaning you’ll see even more time savings as you scale up the number of samples you have.

CUT&Tag delivers:

When to Use CUT&Tag vs CUT&RUN

Both CUT&Tag and CUT&RUN help you unravel protein-DNA interactions when you are short on time and/or sample.

Use the table below to figure out which method is the right one for you!

CUT&Tag is compatible with next-generation sequencing (NGS) and is ideal for studying how histone modifications regulate chromatin binding when sample and time are limited. It can also be used to study transcription factors when the antibody used is validated specifically for CUT&Tag by you or a commercial vendor like CST.

Order the CST^® CUT&Tag Assay Kit #77552 to get all the reagents you need for your CUT&Tag experiment or purchase just the reagents you need a la carte. All CUT&Tag reagents are stringently validated in-house to ensure you’ll get high-quality reagents every time. You can also choose from an ever-expanding list of CST CUT&Tag-validated antibodies.

Comparable Data, Faster Results

CUT&Tag reagents give you the same high-quality data possible with CUT&RUN in half the library prep time.

Analyzing Histone Modifications with CUT&Tag

Generate equivalent data to ChIP-seq and CUT&RUN with the CUT&Tag Assay Kit #77552, CUT&Tag Dual Index Primers and PCR Master Mix for Illumina #47415, or a la carte products when studying histone modifications like Tri-Methyl-Histone H3 (Lys4) or Tri-Methyl-Histone H3 (Lys27). Go from cells to library DNA in 1-2 days with 100,000 starting cells (Figure 2).

Figure 2. ChIP-seq, CUT&RUN, or CUT&Tag assays were performed with HCT 116 cells and Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb #9751, using the SimpleChIP^® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005, CUT&RUN Assay Kit #86652, and CUT&Tag Assay Kit #77552, respectively. DNA libraries were prepared using the DNA Library Prep Kit for Illumina (ChIP-seq, CUT&RUN) #56795 for ChIP-seq and CUT&RUN samples, and the CUT&Tag Dual Index Primers and PCR Master Mix for Illumina #47415 for CUT&Tag samples. The figure shows binding across chromosome 12 (upper), including GAPDH (lower), a known target gene of H3K4me3.

Figure 3. ChIP-seq, CUT&RUN, and CUT&Tag assays were performed with NCCIT cells and Tri-Methyl-Histone H3 (Lys27) (C36B11) Rabbit mAb #9733, using the SimpleChIP^® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005, CUT&RUN Assay Kit #86652, and CUT&Tag Assay Kit #77552. DNA libraries were prepared using DNA Library Prep Kit for Illumina Systems (ChIP-seq, CUT&RUN) #56795 for ChIP-seq and CUT&RUN samples and CUT&Tag Dual Index Primers and PCR Master Mix for Illumina Systems #47415 for CUT&Tag samples. The figures show enrichment of H3K27me3 across chromosome 10 (upper), including the PAX2 gene (lower).

Analyzing Transcription Factors and Cofactors with CUT&Tag

Generate equivalent data to ChIP-seq and CUT&RUN with the CUT&Tag Assay Kit #77552, CUT&Tag Dual Index Primers and PCR Master Mix for Illumina #47415, and CUT&Tag-validated antibodies when studying transcription factors or cofactors like Nanog (Figure 4), Estrogen Receptor α (Figure 5), and JARID2 (Figure 6). Go from cells to library DNA in 1-2 days with 100,000 starting cells.

Figure 4. ChIP-seq, CUT&RUN, or CUT&Tag assays were performed with F9 cells and Nanog (D2A3) XP Rabbit mAb (Mouse Specific) #8822, using the SimpleChIP^® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005, CUT&RUN Assay Kit #86652, and CUT&Tag Assay Kit #77552, respectively. DNA libraries were prepared using the DNA Library Prep Kit for Illumina (ChIP-seq, CUT&RUN) #56795 for ChIP-seq and CUT&RUN samples, and the CUT&Tag Dual Index Primers and PCR Master Mix for Illumina #47415 for CUT&Tag samples. The figure shows binding across chromosome X (upper), including Xist (lower), a known target gene of Nanog.

Figure 5. ChIP-seq, CUT&RUN, or CUT&Tag assays were performed with MCF7 cells grown in phenol red-free medium and 5% charcoal-stripped FBS for 4 days then treated with β-estradiol (10 nM) for 45 minutes and Estrogen Receptor α (D8H8) Rabbit mAb #8644, using the SimpleChIP^® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005, CUT&RUN Assay Kit #86652, and CUT&Tag Assay Kit #77552, respectively. DNA libraries were prepared using the DNA Library Prep Kit for Illumina (ChIP-seq, CUT&RUN) #56795 for ChIP-seq and CUT&RUN samples and the CUT&Tag Dual Index Primers and PCR Master Mix for Illumina #47415 for CUT&Tag samples. The figure shows binding across chromosome 21 (upper), including TFF1 (lower), a known target gene of Estrogen Receptor α.

Figure 6. CUT&Tag, CUT&RUN and ChIP-seq assays were performed with NCCIT cells and JARID2 (D6M9X) Rabbit mAb #13594, using the CUT&Tag Assay Kit #77552, the CUT&RUN Assay Kit #86652, or the SimpleChIP^® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA libraries were prepared using the CUT&Tag Dual Index Primers and PCR Master Mix for Illumina Systems #47415 for CUT&Tag samples and the DNA Library Prep Kit for Illumina Systems (ChIP-seq, CUT&RUN) #56795 for ChIP-seq and CUT&RUN samples. The upper panel compares enrichment around HOXA genes, while the lower panel compares enrichment around HOXD genes, both are known target genes of JARID2.

Analyzing Tissue Samples with CUT&Tag

You can also analyze histone marks in tissue samples using CUT&Tag. We recommend using the CUT&RUN Assay Kit #86652 if you are analyzing transcription factors or cofactors in tissues.

Figure 7. ChIP-seq, CUT&RUN, and CUT&Tag assays were performed with 25 mg (for ChIP-seq) or 1 mg (for CUT&RUN and CUT&Tag) of fresh mouse brain tissue and Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb #9751, using SimpleChIP^® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005, CUT&RUN Assay Kit #86652, or CUT&Tag Assay Kit #77552. DNA libraries were prepared using the DNA Library Prep Kit for Illumina Systems (ChIP-seq, CUT&RUN) #56795 for ChIP-seq and CUT&RUN samples and the CUT&Tag Dual Index Primers and PCR Master Mix for Illumina Systems #47415 for CUT&Tag samples. The figures show enrichment of H3K4me3 across chromosome 6 (upper), including Gapdh (lower), a known target gene of H3K4me3.

Figure 8. ChIP-seq, CUT&RUN, and CUT&Tag assays were performed with 25 mg (for ChIP-seq) or 1 mg (for CUT&RUN and CUT&Tag) of fresh mouse liver tissue and Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb #9751, using SimpleChIP^® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005, CUT&RUN Assay Kit #86652, or CUT&Tag Assay Kit #77552. DNA libraries were prepared using DNA Library Prep Kit for Illumina Systems (ChIP-seq, CUT&RUN) #56795 for ChIP-seq and CUT&RUN samples and CUT&Tag Dual Index Primers and PCR Master Mix for Illumina Systems #47415 for CUT&Tag samples. The figures show enrichment of H3K4me3 across chromosome 6 (upper), including Gapdh (lower), a known target gene of H3K4me3.

Figure 9. ChIP-seq, CUT&RUN, and CUT&Tag assays were performed with 25 mg (for ChIP-seq) or 1 mg (for CUT&RUN and CUT&Tag) of fresh mouse heart tissue and Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb #9751, using SimpleChIP^® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005, CUT&RUN Assay Kit #86652, or CUT&Tag Assay Kit #77552. DNA libraries were prepared using DNA Library Prep Kit for Illumina Systems (ChIP-seq, CUT&RUN) #56795 for ChIP-seq and CUT&RUN samples and CUT&Tag Dual Index Primers and PCR Master Mix for Illumina Systems #47415 for CUT&Tag samples. The figures show enrichment of H3K4me3 across chromosome 6 (upper), including Gapdh (lower), a known target gene of H3K4me3.

Sequencing Success Even When Bioanalyzer or TapeStation System Signal Is Low

Purified CUT&Tag DNA can be quantitated using platforms like the Thermo Fisher Scientific NanoDrop or Qubit Fluorometric Quantification system before being QC’d with platforms like the Agilent Bioanalyzer or TapeStation system prior to NGS.

It should be noted that the calculated DNA library yield may differ depending on the quantitation method used. Check out the CST blog “CUT&Tag DNA Library Yield: What to Do if Yours Is Too Low to Detect With an Agilent Bioanalyzer or TapeStation System” to learn more.

You can still successfully sequence your DNA library even if you see very weak or no visible peaks in the Agilent Bioanalyzer or TapeStation system profile regardless of the CUT&Tag reagents used (Figure 10a) because CUT&Tag baselines are lower than ChIP-seq and CUT&RUN. Therefore, we recommend proceeding with sequencing because it is still possible to obtain sequencing data with high genomics signal even if the Bioanalyzer or TapeStation system signal is low (Figure 10b).

Figure 10a. CUT&Tag was performed with HCT 116 cells and TCF4/TCF7L2 (C48H11) Rabbit mAb #2569, with several sources of loaded pAG-Tn5. The amount of each enzyme used was based on the manufacturer’s recommendation. DNA libraries were prepared using the CUT&Tag Dual Index Primers and PCR Master Mix for Illumina #47415. The figure shows the Bioanalyzer system profile of the CUT&Tag library DNA. The same DNA libraries were sequenced and their NGS tracks are shown in Figure 10b.

Figure 10b. The DNA libraries analyzed with the Bioanalyzer system in Figure 12a were sequenced and NGS tracks are shown. NGS data shows equivalent binding across chromosome 8 (upper), including MYC (lower), a known target gene of TCF4.

Alternatively, you can QC your CUT&Tag DNA library with qPCR by using primers against known positive and negative gene loci on the library DNA to determine the enrichment of chromatin fragments before NGS.