CUT&Tag-IT™ Assay Kit – Tissue
ManualCatalog No. 53170 (16 rxns)
US Pat. No. 10,689,643, EP Pat. No. 2999784
and related patents and applications(version A2)

Copyright 2023 Active Motif, Inc.

Revision

Date

Description of Change

A2

January, 2024

Tagmentation Buffer storage temp changed from
-20ºC to 4°C or -20ºC

Information in this manual is subject to change without notice and does not constitute a commitment on thepart of Active Motif, Inc. It is supplied on an “as is” basis without any warranty of any kind, either explicit or
implied. Information may be changed or updated in this manual at any time. This documentation may not be
copied, transferred, reproduced, disclosed, or duplicated, in whole or in part, without the prior written consent of Active Motif, Inc. This documentation is proprietary information and protected by the copyright laws of
the United States and international treaties. The manufacturer of this documentation is Active Motif, Inc.
US Pat. No. 10,689,643, EP Pat. No. 2999784 and related patents and applications© 2023 Active Motif, Inc., 1914 Palomar Oaks Way, Suite 100; Carlsbad, CA 92008. All rights reserved.
All trademarks, trade names, service marks or logos referenced herein belong to their respective companies.
Oligonucleotide sequences © 2019 Illumina, Inc. All rights reserved.
The guinea pig anti-rabbit secondary antibody is provided under license from Rockland.
pluriStrainer® is a registered trademark of PuriSelect.
For Research Use Only. Not for use in diagnostic procedures. Permitted Use; Resale Prohibited
In the absense of an express written agreement to the contrary, all products are sold and services deliverables are provided by Active Motif for (a) internal in vitro research purposes only and may not be used forservices or any other commercial purpose (b) the exclusive use of the original purchaser, and are not to beresold. You agree not to reverse engineer or otherwise attempt to discover the structure or composition of
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TABLE OF CONTENTS

Page

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1
Kit Components and Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Kit Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Additional Materials Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Flash Freezing Tissue Sample Protocols. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Section A. Prepare Concanavalin A Beads . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Section B. Nuclei Extraction from Tissue Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Section C. Bind Primary Antibody . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Section D. Bind Secondary Antibody. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Section E. Bind CUT&Tag-IT™ Assembled pA-Tn5 Transposomes . . . . . . . . . . . . . . . . . 12
Section F. Tagmentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Section G. DNA Extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Section H. PCR Amplification. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .14
Index Primers and Sample Sheet Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .17
Troubleshooting Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .17
Technical Services . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

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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 interest1.
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 totarget 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 protocolthat are advantageous in certain situations. Instead of the sonication of fixed chromatin andimmunoprecipitation steps performed in ChIP-Seq protocols, in CUT&Tag, fresh or frozen unfixed
cells or tissue 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 sheared 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.
The CUT&Tag-IT™ Assay Kit – Tissue is optimized for 10 mg tissue per reaction. This Kit provides
optimized reagents and protocol to produce 16 unique sequencing-ready Illumina®-compatible
libraries. This kit has been tested with and is recommended for heart, brain, spleen, kidney, andliver tissue.
CUT&Tag-IT™ Assay Kit – Tissue Advantages• Compatible with 2 mg to 30 mg tissue per reaction• Works with heart, brain, spleen, kidney, and liver tissue• Low background signal enables lower sequencing depth• No artifacts caused by formaldehyde crosslinking
product
CUT&Tag-IT™ Assay Kit – Tissue

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formatcatalog no.

16 reactions

53170

Kit Components and Storage
The kit contains sufficient reagents for 16 CUT&Tag Assay reactions. The reagents in this kit havemultiple storage temperatures. Please store components according to the storage conditions below. All reagents are guaranteed stable for 6 months from date of receipt when stored properly.

CUT&Tag Assay Kit – Tissue Reagents
Reagents

Quantity

Storage

5% Digitonin

610 µL-20°C

CUT&Tag-IT™ Lysis Buffer – Tissue

16 mL

RT

16 Strainers

RT

Concanavalin A Beads

320 µL

4°C

CUT&Tag-IT™ Assembled pA-Tn5 Transposomes

16 µL

-20°C

Tagmentation Buffer

2 x 2 mL

4°C or -20ºC

1X Binding Buffer

55 mL

4°C

1X Wash Buffer

40 mL

4°C

Dig-Wash Buffer

50 mL

4°C

Antibody Buffer

800 µL

4°C

Dig-300 Buffer

50 mL

4°C

Guinea Pig Anti-Rabbit Antibody

16 µL

-20°C

Rabbit Anti-Mouse Antibody

16 µL

-20°C

Protease Inhibitor Cocktail

3 x 500 µL-20°C

0.5M EDTA

290 µLRT

10% SDS490 µLRT

10 µg/µL Proteinase K

490 µL-20°C

16 columns

RT

DNA Purification Binding Buffer

50 mL

RT

DNA Purification Wash Buffer

10 mL

RT

DNA Purification Elution Buffer

5 mL

RT

3M Sodium Acetate

500 µLRT

10 mM DNTPs

40 µL

-20°C

5X Q5 Buffer

2 vials of 140 µL

-20°C

8 µL

-20°C

40 µm Strainer

DNA Purification Columns

Q5 High Fidelity DNA Polymerase (2U/µL)

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i5 Indexed Primer 1

10 µL

-20°C

i5 Indexed Primer 2

10 µL

-20°C

i5 Indexed Primer 3

10 µL

-20°C

i5 Indexed Primer 4

10 µL

-20°C

i7 Indexed Primer 1

10 µL

-20°C

i7 Indexed Primer 2

10 µL

-20°C

i7 Indexed Primer 3

10 µL

-20°C

i7 Indexed Primer 4

10 µL

-20°C

SPRI Beads

880 µL

4°C

Additional Materials Required• Mouse or rabbit primary antibody to histone target of interest• Dounce Homogenizer (1 mL, Active Motif Catalog Number 40401 or 15 mL 40415, or similar)• Petri dish to prepare tissue sample in (30 mm to 60 mm diameter)• Forceps to handle tissue• Razor blade to prepare tissue• Scale to weigh tissue sample• Laboratory wipes such as Kimwipes• Dry ice to keep tissue sample and tools chilled before processing• Wet ice to keep tissue sample cool while preparing nuclei• Method to count nuclei such as an automated cell counter or manual hemocytometer with
Trypan Blue. We recommend automated cell counting methods such as the Countess II from
Thermo Fisher Scientific for this assay.
• Molecular grade nuclease free water• 100% Ethanol• 80% Ethanol• Magnetic Rack for 1.5/2 mL tubes• Magnetic Rack for 200 µL 8-well PCR strip tubes• Nutator or orbital rotating mixer• End-over-end rotator• Vortexer• Thermal cycler• Illumina® Sequencer• 1.5 mL Low-bind Microcentrifuge tubes• 2 mL Low-bind Microcentrifuge tubes
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3• Microcentrifuge for 8-well PCR strip tubes with caps, or for 0.2 and 1.5 mL tubes• Multichannel pipette (20 - 200 µL) and 1 mL pipette• Filter tips for pipettes

CUT&Tag Assay Kit – Tissue Protocol
This Kit is compatible with a range of tissue amounts, from 3 mg to 30 mg, and has been optimized for 10 mg tissue sample per reaction of heart, brain, liver, kidney or spleen tissue.
Tissue samples may be fresh or flash frozen on liquid nitrogen or dry ice. To flash freeze tissue
samples for the assay at a later date, follow either the liquid nitrogen or dry ice steps below.
To Flash Feeze on Liquid Nitrogen
1.

Excise the tissue from the animal and place in a microfuge tube.

2.

Submerge in liquid nitrogen for 2 minutes.

3.

Store at -80°C.

To Flash Freeze on Dry Ice
1.

Excise the tissue from the animal and place in a microfuge tube.

2.

Place tube on dry ice with ethanol for 15 minutes.

3.

Store at -80°C.

Section A. Prepare Concanavalin A Beads(30 minutes)
1.

20 µL of resuspended Concanavalin A Bead slurry will be needed per tissue sample. Use
20 µL for up to 30 mg tissue. The kit has been optimized for 10 mg tissue sample reactions.
The following steps are written for 1 sample. The Concanavalin A Beads can be prepared in
one tube as a larger batch for multiple samples. Keep any Concanavalin A Beads not being
used for sample reactions at 4°C after taking the amount needed for the samples planned in
the experiment.

2.

Into a 2 mL microcentrifuge tube, transfer 20 µL Concanavalin A bead slurry into 1.6 mL
1 X Binding Buffer and mix by pipetting. Place the tube on a magnetic stand to clear (30seconds to 2 minutes).

3.

Withdraw the liquid completely, avoiding the beads, and remove the tube from the magnetic stand. Add 1.5 mL 1 X Binding Buffer to the tube, mix by pipetting, and collect any
liquid from the sides or cap with a quick pulse spin in a microcentrifuge.

4.

Place tube on magnetic stand to clear (30 seconds to 2 minutes), and remove liquid by pipette, avoiding the beads. Resuspend in 20 µL 1 X Binding Buffer (20 µL per 10 mg sample)
and keep the tube containing the prepared Concanavalin A Beads at room temperature until
the tissue samples are ready.
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4Section B. Nuclei Extraction from Tissue Samples
FIRST:

Prepare CUT&Tag-IT™ Lysis Buffer – Tissue with fresh Protease Inhibitor Cocktail.
Per sample, add 10 µL Protease Inhibitor Cocktail to 990 µL CUT&Tag-IT™ Lysis
Buffer – Tissue for 1 mL total volume. Keep the freshly prepared buffer on wet
ice until it is used in the steps below.
Prepare 1 X Wash Buffer with fresh Protease Inhibitor Cocktail. Per sample,
add 7 µL Protease Inhibitor Cocktail to 743 µL 1 X Wash Buffer for 750 µL total
volume. Keep the freshly prepared buffer on wet ice until it is used in the steps
below.

5.

Place Dounce Homogenizer on wet ice to chill.

6.

Place petri dish on scale and tare to be ready to weigh tissue sample.

7.

Place the petri dish on dry ice to be ready for the tissue sample.

8.

Ethanol spray the forceps and place on dry ice to chill.

9.

Ethanol spray the razor blade and carefully wipe dry with clean laboratory wipe.

10. Place tissue in petri dish and place on scale to weigh. Decide how much to cut for a 10 mg
piece per assay reaction. If multiple samples of the same tissue will be assayed, the tissue
can be processed in bulk. For example, if 4 reactions of the same tissue will be assayed, all
40 mg of the tissue can be processed in one petri dish.
a.

To cut tissue to desired size, place petri dish with tissue back on dry ice.

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11.

b.

Hold petri dish firmly and use razor to chop the tissue to smaller pieces. This is to get
10 mg per sample for the reaction. The finer chopping for tissue lysis is in step 12
below.

c.

Place dish with tissue section on scale to check weight, and repeat adding or dividing
sample and chopping until target weight is reached.

Place petri dish with tissue sample on wet ice. Add 1 mL of the freshly prepared CUT&Tag-IT
Lysis Buffer – Tissue to the 10 mg sample. Lift one side of the petri dish so the tissue andbuffer run down to the far side, while the tissue remains covered with buffer. Let sit 1
minute. If processing a larger amount of tissue for multiple reactions, add an adequate
amount of CUT&Tag-IT Lysis Buffer to sufficiently cover the tissue as it is processed.

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612. Partially lift the petri dish so that the tissue and the buffer run down to the corner of the dishso the tissue stays covered in the CUT&Tag-IT™ Lysis Buffer – Tissue and can be minced into
smaller pieces. Using the razor blade, start slicing firmly through the tissue.

13. Lay the petri dish flat and mince all the larger pieces until no chunks of tissue can be seen.
Chop until tissue pieces can no longer be chopped into smaller pieces by the razor. Mince
until the pieces are small enough that they can be easily aspirated by a P1000 pipette.

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714. Using the razor blade, push the finely minced tissue in the CUT&Tag-IT™ Lysis Buffer to anedge of the dish. This will make the sample easier to collect.
15. With clean and disinfected scissors, cut the narrow tip off of a 1 mL pipette tip. Use this topipette the sample out of the petri dish and into the Dounce Homogenizer.

16. Dounce sample with tight pestle 30 times, being careful not to move the pestle above themeniscus to prevent forming bubbles.
If the pestle is very tight and very difficult to move, first do 20 strokes with the loose pestle,
then change to the tighter pestle for 10 strokes.
17.

Place a 40 µm Strainer into a microcentrifuge tube on wet ice. One 40 µm Strainer and one
corresponding microcentrifuge tube will be needed per 1 mL of sample. These Strainers fit
1.5 ml, 2ml microcentrifuge tubes and 15 ml conical tubes.

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818. Pipette the Dounced tissue into the 40 µm Strainer on the microcentrifuge tube. Be careful
to avoid large tissue pieces so the mesh does not get clogged. Gently move the 40 µm
Strainer up and down inside the tube to help the sample flow through. Hold the tube with
one hand and move the Strainer up and down with the other hand without fully removing
the Strainer from the tube to create a void for the lysed tissue to flow into. Once the sample
has moved through the Strainer, discard the Strainer.

19. Centrifuge sample tubes at 500 X g for 5 minutes at 4°C to pellet nuclei.
20. Remove supernatant from sample and discard.
21. Resuspend pelleted nuclei in 375 µL of ice cold 1 X Wash Buffer per 1.5 ml microcentrifuge
tube per sample reaction being assayed. This is the nuclei suspension. If there are multiple
reactions of the same sample type, pool them together to count nuclei.
NOTE:

The number of nuclei per mg will vary by tissue type. We observed an averageof ~150,000 nuclei per mg. Counting accuracy is critical and we recommend an
automated counting method versus a hemocytometer.

22. Take a sample of the nuclei suspension and count. Calculate nuclei density and normalize
samples to a concentration of 250,000 nuclei in 375 µL 1 X Wash Buffer per sample. Add
freshly prepared 1 X Wash Buffer if needed to obtain that concentration.
NOTE:

If you are normalizing to use the same amount of nuclei per reaction, we recommend CUT&Tag reactions in the range of 100,000 to 500,000 nuclei.

NOTE:

If you have excess nuclei, you may store them in a separate cryo-vial. Add
DMSO to 10% of the total volume in the vial. Freeze these samples overnightin an isopropanol freezing container in a -80°C freezer. If you are planning on
storing nuclei for more than 1 month, transfer these samples to a liquid nitrogencontainer.

23. Add 20 µL of the Concanavalin A Beads that were prepared in Section A to each sample.
Incubate on an end-over-end (orbital) rotator for 10 minutes at room temperature.
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9Section C. Bind Primary Antibody (2 hr to Overnight)
FIRST:

Before using Antibody Buffer in Step 26 below, you must add Protease Inhibitor
Cocktail and Digitonin. For every 1 mL of buffer to be used, add 10 µL of Protease Inhibitor Cocktail and 10 µL of 5% Digitonin. Keep on ice. Antibody Buffer
can be kept at 4°C for up to 2 days once Protease Inhibitor Cocktail and Digitonin have been added. For each reaction, you will need 50 µL supplemented
with 0.5 µL Protease Inhibitor Cocktail and 0.5 µL of 5% Digitonin.

24. After a quick spin (<100 X g) to remove liquid from the cap, place the tubes on a magnetic
stand to clear (30 seconds to 2 minutes).
25. Place new microcentrifuge tubes (one for each sample) on ice. Remove the supernatantfrom the tubes in the step above via a pipette and transfer to new tubes. There will beapproximately 395 µL each. These tubes contain the unbound nuclei and are not an assay
reaction. Count the number of unbound nuclei via a hemocytometer.
26. Calculate the number of bound nuclei by subtracting the number of unbound nuclei fromthe total per reaction in step 22. This is a check to be certain nuclei are bound for the assay reactions. We typically observed a range of 0 - 4938 unbound nuclei per reactions of
250,000 - 500,000 nuclei. Resuspend bound nuclei from the tubes with the beads in step
25 in a volume of freshly prepared ice-cold Antibody Buffer such that each sample is in a 50
µL volume, with a minimum of 100,000 nuclei per 50 µL. Gently vortex. Place the tube on
ice.
27. Add 1 µL (or at least 1 µg) undiluted primary antibody to each sample, and gently vortex or
pipette to mix.
IMPORTANT: Use a rabbit or mouse primary antibody. We recommend using a 1:50 to
1:100 dilution or the antibody manufacturer’s recommendation for the dilution
used in applications such as immunofluorescence. Histone H3K27me3 (Cat. No.
39157) is a common mark that can be a positive control.
28. Incubate overnight at 4°C with orbital mixing, make certain liquid stays together in the bottom of the tube so that it all mixes together.

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10Section D. Bind Secondary Antibody (1.5 hours)
FIRST:

Before using Dig-Wash Buffer in Step 30 below, you must add Protease Inhibitor
Cocktail and Digitonin. For every 1 mL of buffer, add 10 µL Protease Inhibitor
Cocktail and 10 µL of 5% Digitonin. 3.1 mL total are used per sample. Keep on
ice. Dig-Wash Buffer can be kept at 4°C for up to 2 days once Protease Inhibitor
Cocktail and Digitonin have been added.

IMPORTANT:

Choose the right secondary antibody for the reactions!

If a mouse primary antibody was used, then use the Rabbit Anti-Mouse Secondary Antibody.
If a rabbit primary antibody was used, then use the Guniea Pig Anti-Rabbit
Secondary Antibody.
29. After a quick spin (<100 X g), place each tube on a magnetic stand to clear (30 seconds to 2
minutes), and then remove the liquid via a pipette.
30. Dilute the Secondary Antibody 1:100 in Dig-Wash Buffer. 100 µL of the diluted secondary areneeded per reaction. Add 100 µL of this diluted secondary antibody to each sample (making certain that the correct Secondary Antibody is used per primary antibody) while gently
vortexing to dislodge the beads from the sides of the tube.
31. Place the tubes on an orbital rotator at room temperature for 60 minutes.
32. After a quick spin, place the tubes on a magnetic stand to clear (30 seconds to 2 minutes),then remove the liquid via a pipette.
33. Add 1 mL Dig-Wash Buffer to each reaction. Gently vortex or pipette to dislodge any beads
that have aggregated together.
34. Repeat Steps 32-33 twice, for a total of three washes.

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11Section E. Bind CUT&Tag-IT™Assembled pA-Tn5 Transposomes(1.5 hours)
FIRST:

Before using Dig-300 Buffer in Step 35 below, you must add Protease Inhibitor
Cocktail and Digitonin. For every 1 mL of buffer, add 10 µL Protease Inhibitor
Cocktail and 2 µL of 5% Digitonin. 3.1 mL total are used per sample. Keep on
ice. Dig-300 Buffer can be kept at 4°C for up to 2 days once Protease Inhibitor
Cocktail and Digitonin have been added.

35. Dilute the CUT&Tag-IT™ Assembled pA-Tn5 Transposomes with Dig-300 Buffer to a final
concentration of 1:100. 100 µL will be needed per reaction. For example, for one reaction,
add 1 µL of the CUT&Tag-IT™ Assembled pA-Tn5 Transposomes to 100 µL of Dig-300 Buffer.
36. After a quick spin (<100 X g), place each tube containing the immunoprecipitated samples
on a magnetic stand to clear (30 seconds to 2 minutes), and then remove the liquid via a
pipette.
37. Add 100 µL of the diluted CUT&Tag-IT™ Assembled pA-Tn5 Transposomes from Step 35 whilegently pipetting to make sure it mixes together.
38. Incubate the reactions at room temperature on an orbital rotator for 60 minutes.
39. After a quick spin (<100 X g), place the tubes on a magnetic stand to clear (30 seconds to 2
minutes) and remove the liquid with a pipette.
40. Add 1 mL Dig-300 Buffer. Gently vortex or pipette to dislodge the beads.
41. Repeat Step 39-40 twice for a total of three washes.

Section F. Tagmentation (60 minutes)
FIRST:

Before using Tagmentation Buffer in Step 43 below, you must add Protease
Inhibitor Cocktail and Digitonin. For every 1 mL of Tagmentation Buffer to be
used, add 10 µL Protease Inhibitor Cocktail and 2 µL of 5% Digitonin.

42. After a quick spin (<100 X g), place the tubes on a magnetic stand (30 seconds to 2 minutes)
and remove the liquid with a pipette.
43. Add 125 µL Tagmentation Buffer while gently vortexing or pipetting to mix each tube.
44. Incubate tubes at 37°C for 60 minutes.

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12Section G. DNA Extraction (60 minutes)
45. To stop tagmentation and solubilize DNA fragments, add to each sample:
4.2 µL 0.5 M EDTA
1.25 µL 10% SDS
1.1 µL Proteinase K (10 mg/mL)
46. Mix by vortexing at full speed briefly for approximately two seconds, and incubate for 60
minutes at 55°C to digest.
NOTE:

It is typical for the beads to form a large clump during incubation with Proteinase K and SDS due to the viscoelasticity of DNA. However, for abundant
genome-wide epitopes, large-scale fragmentation of the genome will normally
result in reduced clumping and release of beads into suspension, turning theliquid brownish relative to negative controls.

47. After a quick spin (<100 X g), place the tubes on a magnetic stand (30 seconds to 2 minutes)
and transfer the liquid with a pipette into a new 1.5 mL microcentrifuge tube.
48. Add 625 µL DNA Purification Binding Buffer to each sample and mix by pipetting up anddown. If the color indicator turns violet or orange, add 8 µL of 3 M Sodium Acetate.
49. For each sample, place a labeled DNA Purification Column into a collection tube.
50. Transfer each sample to its corresponding column, close the cap, and centrifuge at 17,000 X
g (~14,000 rpm) for 1 minute.
51. Discard the flow-through and return the column to the collection tube.
NOTE:

100% Ethanol must be added to the DNA Purification Wash Buffer before thefirst use, to a final concentration of 80% (40 mL of 100% Ethanol to the DNA
Purification Wash Buffer bottle).

52. Add 750 µL of DNA Purification Wash Buffer (prepared above) to the column and cap thecolumn. Centrifuge at 17,000 X g for 1 minute.
53. Discard the flow-through and return the collection tube to the column. Centrifuge the empty
tube at 17,000 X g for 2 minutes to remove any remaining DNA Purification Wash Buffer.
54. Transfer each column to a new microcentrifuge tube and add 35 µL of DNA Purification
Elution Buffer to the center of the column matrix, cap the column, and incubate at room
temperature for 1 minute.
55. Centrifuge at 17,000 X g for 1 minute. Discard the column. The DNA purification is complete,
and the DNA can be stored at -20°C or you may proceed to the PCR Amplification steps
below.

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13Section H. PCR Amplification
56.

Set up the PCR reactions by adding the components below in the order shown. If libraries
are to be multiplexed for sequencing, ensure that a unique i5 and i7 index combination isused for each.
Each sample will require a combination of one i7 Indexed Primer and one i5 Indexed Primer
in the PCR amplification reaction. There are 4 x 4 = 16 unique combinations of i7/i5 primers
for a total of 16 samples that can be multiplexed. These Indexed Primers are based on
Illumina’s Nextera adapters.
Per reaction:
Use one i7 Indexed Primer

And use one i5 Indexed Primer

i7 Indexed Primer 1 = i7 N701

i5 Indexed Primer 1 = i5 N501

i7 Indexed Primer 2 = i7 N702

i5 Indexed Primer 2 = i5 N502

i7 Indexed Primer 3 = i7 N703

i5 Indexed Primer 3 = i5 N503

i7 Indexed Primer 4 = i7 N704

i5 Indexed Primer 4 = i5 N504

Reagent per CUT&TAG sample

Volume (50 µL)

Tagmented DNA

30 µL

i7 Indexing Primer

2.5 µLi5 Indexing Primer

2.5 µLdNTPs (10 mM)

1.0 µL5X Q5 Buffer

10 µL

Molecular grade nuclease-free water

3.5 µL

Q5 High-Fidelity DNA Polymerase

0.5 µL(Total Volume = 50 µL)

57. Perform PCR using the following program on a thermal cycler with a heated lid:
72°C for 5 minutes
98°C for 30 seconds
12 cycles of: 98°C for 10 seconds, 63°C for 10 seconds
72°C for 1 minute (this step follows the 12 cycles and is not part of the cycling above)
Hold at 10 °C

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1458. Perform SPRI Bead clean-up following the steps below, per sample you will need 55 µL SPRI
Beads (1.1 X sample volume), eluting in 20 µL DNA Purification Elution Buffer. 400 µL freshlyprepared 80% Ethanol will also be required per sample.
a.

Add 60 µL well-mixed, room temperature SPRI Beads to each sample.

b.

Vortex briefly to mix, and incubate for 5 minutes at room temperature to allow beads
to bind.

c.

Apply magnet to collect beads.

d.

Once the solution is clear, aspirate the supernatant.

e.

With the magnet still applied to the sample, add 180 µL 80% Ethanol to each sample
without mixing.

f.

Incubate for 30 seconds at room temperature.

g.

Aspirate the supernatant.

h.

Repeat steps e through g for a second ethanol wash.

i.

Allow tubes to sit at room temperature with lids open so that residual ethanol canevaporate. Oncethe beads transition from shiny to matte (2 - 5 minutes), proceed to the next step.

j.

With the tubes separated from the magnet, add 20 µL DNA Purification Buffer.

k.

Cap tubes and vortex to mix.

l.

Incubate samples for 5 minutes at room temperature.

m.

Apply magnet to sample and allow beads to pellet.

n.

Once the solution is clear, transfer each supernatant, containing elute DNA to a fresh,clean tube.

59. At this stage, libraries are ready for quantification and sequencing.

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Index Primers and Sample Sheet Information
Index 1 (i7) Primers
CAAGCAGAAGACGGCATACGAGAT[i7]GTCTCGTGGGCTCGG
Index 2 (i5) Primers
AATGATACGGCGACCACCGAGATCTACAC[i5]TCGTCGGCAGCGTC
i7 Indexi7 Sequence

Sample SheetN701

TCGCCTTATAAGGCGA

N702

CTAGTACGCGTACTAG

N703

TTCTGCCTAGGCAGAA

N704

GCTCAGGA

TCCTGAGC

i5 Indexi5 Sequence

Sample Sheet (NovaSeq
v1.0 Reagent Kits, MiSeq,
HiSeq 2000/2500)

N501

TAGATCGCTAGATCGC

N502

CTCTCTATCTCTCTAT

N503

TATCCTCTTATCCTCT

N504

AGAGTAGAAGAGTAGA

i5 Indexi5 Sequence

Sample Sheet (NovaSeq
v1.5 Reagent Kits iSeq,
MiniSeq, NextSeq,
HiSeq 3000/4000)

N501

TAGATCGCGCGATCTA

N502

CTCTCTATATAGAGAG

N503

TATCCTCTAGAGGATA

N504

AGAGTAGATCTACTCT

Sequence for Read 1 and Read 2 adapter trimming: CTGTCTCTTATACACATCT.
NOTE:

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2 Million to 10 Million sequencing reads are recommended. 10 Million reads
typically yield 20,000 peaks in our quality control testing.

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References1.

Kaya-Okur, H.S. et al. (2019) Nature comm. 10:1930 (1).

Troubleshooting Guide - See More FAQs on the Kit Web Page
Problem/question

Recommendation

What controls arerecommended for
CUT&Tag?

A good technical positive control for the reagents and workflow is the antibody H3K27me3 (Cat. No. 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 todetermine 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 wasshown to be specific. However, if you wish to add an IgG control this is fine.

Are there any QC steps
recommended?

After library generation, successful library prep quality should be assessedby evaluating the library on a TapeStation or Bioanalyzer. An ideal librarywould 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 havebeen 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 towork with CUT&Tag?

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.

Is CUT&Tag compatible
with both monoclonal
and polyclonal antibodies?

Yes. Please be sure to use the proper species-specific secondary antibody.

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.

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Problem/question

Recommendation

Can more than 16
samples be multiplexed?

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 useddirectly 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 specificgene problematic since it is likely that you will be missing one or the otherprimer 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 containmolecular identifiers?

No. CUT&Tag-IT Assay Kit libraries do not contain molecular identifiers.

Should the CUT&Tag-IT
Assay Kit libraries besequenced as singleend or paired-end?

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

What read length isrecommended 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 datafrom sequencing thesamples generated by
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.

No library visible

This is not uncommon with CUT&Tag libraries, especially for transcription
factors. Libraries that are barely visible still have been shown to sequencewell. qPCR analysis for library abundance (like KAPA Library Quantification)
can help measure how much library is there. If your positive control works,
these are still worth sequencing. If your positive control does not work, you
may have lost nuclei during the protocol. If this may be the case, count cells
bound to beads at the end of Section B to be sure cells are not lost.

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Technical Services
If you need assistance at any time, please call Active Motif Technical Service at one of the numbers listed below.
Active Motif North America
Toll free:Direct:Fax:
E-mail:Active Motif Europe
UK Free Phone:France Free Phone:
Germany Free Phone:Direct:Fax:
E-mail:877.222.9543
760.431.1263
760.431.1351
tech_service@activemotif.com
0800/169 31 47
0800/90 99 79
0800/181 99 10+32 (0)2 653 0001
+32 (0)2 653 0050
eurotech@activemotif.com

Active Motif Japan
Direct: +81 (0)3 5225 3638
Fax: +81 (0)3 5261 8733
E-mail: japantech@activemotif.com
Active Motif China
Direct: (86)-21-20926090
Cell Phone: 18521362870
E-mail: techchina@activemotif.com

Visit Active Motif online at activemotif.com

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