CUT&Tag-IT™ Assay Kit Overview
CUT&Tag vs. CUT&RUN vs. 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.
CUT&Tag-IT™ Assay Kit Data
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. 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 our Nextera™-Compatible Multiplex Primers (96 plex) kit (Cat. No. 53155) enables multiplexing up to 96 reactions. These primers are provided at a concentration of 25 µM to be used directly in our Kits. You could also purchase and combine other 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
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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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|CUT&Tag-IT™ Assay Kit, Anti-Rabbit||16 rxns||53160||$1,200||Buy|
|CUT&Tag-IT™ Assay Kit, Anti-Mouse||16 rxns||53165||$1,200||Buy|