CUT&Tag-IT™ Assay Kit

Rapid and robust genome-wide analysis of histone marks at lower sequencing depths

 

CUT&Tag-IT™ Assay Kit Overview

Cleavage Under Targets and Tagmentation (CUT&Tag) is a method to investigate genomic localization of histone modifications and some transcription factors that reveals interactions between proteins and DNA or identifies DNA binding sites for proteins of interest.

Unlike MNase-Seq or ATAC-Seq methods that target open chromatin and are therefore dependent on chromatin accessibility, CUT&Tag utilizes an antibody-based enzyme tethering strategy to target specific histone modifications or proteins to reveal chromatin-binding information that is specific to those sites or proteins of interest.

CUT&Tag is based on the same principles as ChIP-Seq, but with several changes to the protocol that are advantageous in certain situations. Instead of the sonication of fixed chromatin and immunoprecipitation steps performed in ChIP-Seq protocols, in CUT&Tag, fresh (not frozen) unfixed cells are bound to concanavalin A beads and the antibody incubation is performed with cells in their native state. Directly following antibody binding, the chromatin is digested and NGS libraries are prepared in a single step by tagmentation using the protein A-Tn5 (pA-Tn5) transposome enzyme that has been pre-loaded with sequencing adapters.

CUT&Tag can rapidly produce high-quality results from less starting material than ChIP-Seq, and enables robust analysis from lower sequencing depths, saving both time and money.

Learn more about CUT&Tag and how it works

CUT&Tag-IT™ Assay Kit Advantages:

  • Compatible with as few as 5,000 cells
  • Complete kit with optimized protocol
  • Developed for histone marks and some transcription factors
  • Sequencing-ready libraries without the laborious and costly steps of ChIP-Seq
  • Low background signal enables lower sequencing depth
  • No artifacts caused by formaldehyde crosslinking

How the CUT&Tag-IT™ Assay Works

How the Cut&Tag-IT™ Assay Works

FREE Antibody with Cut&Tag-IT™ Assay Kit Purchase

CUT&Tag vs. CUT&RUN vs. ChIP-Seq

  CUT&Tag CUT&RUN ChIP-Seq
Performed Under Native Conditions? Yes Yes No
Chromatin Fragmentation Method Tn5-based tagmentation MNase digestion Sonication
Cell Number Requirements 5,000-500,000 cells 500,000 cells 1-10 million cells
Sequencing Depth Required * 2 million reads ** 8 million reads 20-50 million reads
Integrated Library Preparation? Yes, uses tagmentation No, separate library prep required No, separate library prep required
Compatible Targets Primarily histone modifications, some transcription factors and co-factors Wide range of histone modifications, transcription factors, and co-factors Wide range of histone modifications, transcription factors, and co-factors
Workflow Length 1-2 days 1-2 days 2-3 days

* Kaya-Okur et al. Nature Communications (2019) 10:1930

** For less abundant targets of interest, 8 to 10 million reads are recommended

 

CUT&Tag-IT™ Assay Kit Contents

The CUT&Tag-IT™ Assay Kit components are shipped at two temperatures, with one box on dry ice for components to be stored at -20°C, and a second box at room temperature for components that are not to be frozen and must be stored at 4°C. Please store components according to the storage conditions in the manual after first use. All reagents are guaranteed stable for 6 months from date of receipt when stored properly.

Reagents included:

  • 5% Digitonin, store at -20°C
  • Concanavalin A Beads, store at 4°C
  • CUT&Tag-IT™ Assembled pA-Tn5 Transposomes, store at -20°C
  • Tagmentation Buffer, store at -20°C
  • 1X Binding Buffer, store at 4°C
  • 1X Wash buffer, store at 4°C
  • Dig-Wash Buffer, store at 4°C
  • Antibody Buffer, store at 4°C
  • Dig-300 Buffer, store at 4°C
  • Guinea Pig Anti-Rabbit Antibody, store at -20°C
  • Protease Inhibitor Cocktail, store at -20°C
  • 0.5M EDTA, store at RT
  • 10% SDS, store at RT
  • 10 µg/µL Proteinase K, store at -20°C
  • DNA Purification Columns, store at RT
  • DNA Purification Binding Buffer, store at RT
  • DNA Purification Wash Buffer, store at RT
  • DNA Purification Elution Buffer, store at RT
  • 3M Sodium Acetate, store at RT
  • 10 mM DNTPs, store at -20°C
  • 5X Q5 Buffer, store at -20°C
  • Q5 High Fidelity DNA Polymerase (2U/µL), store at -20°C
  • i5 Indexed Primer 1, store at -20°C
  • i5 Indexed Primer 2, store at -20°C
  • i5 Indexed Primer 3, store at -20°C
  • i5 Indexed Primer 4, store at -20°C
  • i7 Indexed Primer 1, store at -20°C
  • i7 Indexed Primer 2, store at -20°C
  • i7 Indexed Primer 3, store at -20°C
  • i7 Indexed Primer 4, store at -20°C
  • SPRI Beads, store at 4°C

Don’t forget to use an antibody validated for CUT&Tag!

Active Motif specializes in manufacturing high-quality antibodies to histones, histone modifications, chromatin proteins, and other factors, including a growing list of antibodies that we have experimentally validated in-house to work well in CUT&Tag assays. Check out our list of CUT&Tag-validated antibodies.


For a limited time, you can get a free antibody of your choice with every purchase of the CUT&Tag-IT™ Assay Kit. Learn more.

 

CUT&Tag-IT™ Assay Kit Data

CUT&Tag-IT™ Assay Kit compared with published data(Click image to enlarge)

Figure 1: CUT&Tag-IT Assay Kit data correlate well with ENCODE ChIP-Seq data.
Chromatin landscapes from human K562 cells are shown across a 868 kb segment of the human genome region (Chr16: 10,400-11,250) for H3K27me3 (dark blue), phospho-Pol2Ser2 (red), CTCF (orange), H3K4me3 (green), H3K9ac (light blue) H3K9me3 (purple), and H3K27ac (black) showing high concordance of CUT&Tag-IT Assay Kit signals from 100,000 cells compared with ENCODE ChIP-Seq data from at least one million cells. Antibodies used in the CUT&Tag assays for the following targets were: H3K27me3: cat. no 39155, CTCF: cat. no. 61311, H3K4me3: cat. no. 39159, H3K9ac: cat. no. 39917, H3K9me3: cat. no. 39161, H3K27ac: cat. no. 39133.

 

CUT&Tag-IT™ Assay Kit FAQs

Does the CUT&Tag-IT™ Assay Kit include everything required to perform CUT&Tag?

Yes, the kit contains everything required including the assembled pA-Tn5 transposomes. The only additional material required would be the standard lab equipment and reagents which are listed under Additional Materials Required in the manual.

How is CUT&Tag different from ChIP-Seq?

CUT&Tag differs from ChIP-Seq in the following ways:

  • No fixation is required for CUT&Tag
  • No sonication or fragmentation required for CUT&Tag
  • Lower cell number
  • Integrated library prep
  • Lower background
  • Lower sequencing depth

When should a researcher choose CUT&Tag over ChIP-Seq?

CUT&Tag is a great choice for researchers who have limited cell numbers and are interested in understanding genome-wide changes in histone modifications. For researchers interested in understanding genome-wide transcription factor recruitment (endogenous or tagged) we recommend ChIP-Seq.

What types of samples does the CUT&Tag-IT™ Assay Kit work with?

Currently we have a protocol for cultured cells only.

How many cells are required for the CUT&Tag-IT™ Assay Kit?

We recommend 50,000 – 100,000 cells per reaction. However, we have acquired useful data from as few as 5,000 cells.

How do I prepare my cells for the CUT&Tag-IT™ Assay Kit?

Follow the protocol in the CUT&Tag-IT™ Assay Kit manual. For adherent cells it is critical that trypsin NOT be used to detach cells as it will damage the epitopes for binding to concanavalin A beads. Use an enzyme-free dissociation method instead, such as scraping or rubber policeman.

Can I use the CUT&Tag-IT™ Assay Kit with frozen cells?

Cells must be cryopreserved. Flash frozen cell pellets are not compatible with the protocol. To cryopreserve cells we recommend following our Services sample prep protocol.

What controls are recommended to use with the CUT&Tag-IT™ Assay Kit?

A good technical positive control for the reagents and workflow is the antibody H3K27me3 (cat# 39157). For a negative control we recommend using the secondary antibody without the primary antibody. This will show background from the secondary antibody and pA-Tn5 with your samples.

Do I need to include an IgG control?

The purpose of including an IgG control in CUT&Tag experiments is to determine if the pA-Tn5 is specific to genomic regions where the antibody is located/enriched. This negative control is not used in analysis and is different from the INPUT control used in ChIP-Seq. Active Motif R&D found that adding an IgG control does not add any additional value as the pA-Tn5 was shown to be specific. However, if you wish to add an IgG control this is fine.

Are there any QC steps recommended for the CUT&Tag-IT™ Assay Kit?

After library generation, successful library prep quality should be assessed by evaluating the library on a TapeStation or Bioanalyzer. An ideal library would have most of the fragments below 500 bp. We recommend determining the library concentration by using a KAPA Library Quantification Kit.

Does CUT&Tag require an INPUT control like ChIP-Seq?

No. CUT&Tag does not require the use of an input control.

What antibodies have been validated using the CUT&Tag-IT™ Assay Kit?

For a full list of CUT&Tag-IT™ Assay Kit validated Active Motif antibodies visit: https://www.activemotif.com/catalog/1319/cut-tag-validated-antibodies

Are ChIP-Seq validated antibodies going to work with the CUT&Tag-IT™ Assay Kit?

CUT&Tag and ChIP-Seq have quite different workflows. If an antibody works for ChIP-Seq, that does not necessarily mean it will work in CUT&Tag. We advise using one our CUT&Tag-IT™ Assay Kit validated Active Motif antibodies, or validating a ChIP grade or ChIP-validated antibody.

Is the the CUT&Tag-IT™ Assay Kit compatible with both monoclonal and polyclonal antibodies?

Yes, it is compatible with rabbit derived antibodies.

Are tagged proteins compatible with CUT&Tag?

We have not validated the CUT&Tag-IT™ Assay Kit for tagged proteins. However, in principle, there is no reason why it couldn’t work.

Can the standard Active Motif Spike-In Normalization be used for the CUT&Tag-IT™ Assay Kit?

No. Our standard ChIP-Seq Spike-In Normalization method is not compatible with the CUT&Tag-IT™ Assay Kit.

Can I multiplex more than 16 samples using the CUT&Tag-IT™ Assay Kit?

The CUT&Tag-IT™ Assay Kit is supplied with 4x4 unique dual indexes for 16 unique samples. We do not currently have other index primers available. However, the indexed primers in the kit are identical to the Illumina Nextera primers corresponding to N701-N704 and N501-N504. If you would like to multiplex more than 16 samples you could purchase and combine Illumina Nextera primers at the same concentration (25 μM) as those in the kit.

Is the CUT&Tag-IT™ Assay Kit compatible with qPCR analysis before sequencing?

Yes and no. Yes, you can do qPCR, but because of the randomness of Tn5’s insertion of the adapter it makes designing primers that will prime a specific gene problematic since it is likely that you will be missing one or the other primer landing site. Therefore, qPCR may contain errors as fragments that do not have the landing site will not be measured.

Are the CUT&Tag-IT™ Assay Kit libraries single or dual indexed?

CUT&Tag libraries are dual-indexed libraries.

Do the CUT&Tag-IT™ Assay Kit libraries contain a Molecular Identifiers?

No. CUT&Tag libraries do not contain molecular identifiers.

Should the CUT&Tag-IT™ Assay Kit libraries be sequenced as single-end or paired-end?

The CUT&TAG-IT™ Assay Kit libraries should be sequenced as paired end.

What read length is recommended for the CUT&Tag-IT™ Assay Kit?

We recommend a read length of 2x38 (PE38). This is shorter than the read length described in the Henikoff paper. However, we have not seen an impact on data quality or mapping rates. You can use a higher read length if you wish to.

How do I analyze the sequencing data after using the CUT&Tag-IT™ Assay Kit?

CUT&Tag data is analyzed similarly to ChIP-Seq data. We use a BWA algorithm with peak calling performed using MACS2.

 

 

CUT&Tag-IT™ Assay Kit Documents

 

 

 

Order CUT&Tag-IT™ Assay Kit

 

We recommend using a CUT&Tag-validated antibody for best results. For a limited time, you can get a free antibody of your choice with every purchase of the CUT&Tag-IT™ Assay Kit. Learn more.



You might also be interested in:

US Pat. No. 10,689,643, EP Pat. No. 2999784 and related patents and applications

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