Enabling Epigenetics Research

CUT&Tag-IT™ R-loop Assay Kit
Catalog No. 53167(Version A4)

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

A3

February, 2024

Updated table in Index Primers and Sample Sheet Information on
page 10

A4

May, 2024

Added notes to Cell Preparation on page 4

Information in this manual is subject to change without notice and does not constitute a
commitment on the part 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 inthis 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 andinternational treaties.
The manufacturer of this documentation is Active Motif, Inc.
© 2023 Active Motif, Inc., 1914 Palomar Oaks Way, Suite 150; Carlsbad, CA 92008. All
rights reserved.
All trademarks, trade names, service marks or logos referenced herein belong to theirrespective companies.
US Pat. No. 10,689,643, EP Pat. No. 2999784 and related patents and applications.
For research use only. Not for use in diagnostic procedures.

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Contents
Overview  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Kit Components and Storage  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  2
Additional Materials Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  3
CUT&Tag-IT R-loop Assay Kit Protocol  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  4
Day 1 Binding Cells to Concanavalin A Beads (approximately 2 hours) . . . . . . . . .  4
Prepare Buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  4
Prepare Cells (10 minutes) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  4
Prepare Concanavalin A Beads (20 - 30 minutes) . . . . . . . . . . . . . . . . . . . . . . . .  4
DNA-RNA Hybrid (Clone S9.6) Antibody Binding (overnight) . . . . . . . . . . . . . . .  5
Day 2 (approximately 6 hours) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  6
Bind Secondary Antibody  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  6
Bind CUT&Tag-IT Assembled pA-Tn5 Transposomes (1 hour)  . . . . . . . . . . . . . .  7
Tagmentation (1 hour) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  7
DNA Extraction (1 hour)  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  7
PCR Amplification (1 hour) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  9
Index Primers and Sample Sheet Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Troubleshooting Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Technical Services . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

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Overview
CUT&Tag (Cleavage Under Targets & Tagmentation) is a genome-wide chromatin profilingtechnique that utilizes an antibody-based enzyme tethering strategy to target specifichistone modifications or proteins to reveal chromatin-binding information that is specific tothose sites or proteins of interest. Following antibody binding, NGS libraries are preparedby tagmentation using the protein A-Tn5 (pA-Tn5) transposome enzyme that has been preloaded with sequencing adapters.
This CUT&Tag-IT™ R-loop Assay Kit utilizes a CUT&Tag-based approach to profile R-loops
genome-wide with a specifically optimized protocol for cell samples and our DNA-RNA hybrid(Clone S9.6) antibody.
R-loops are three-stranded nucleic acid structures that are formed when a single-stranded
DNA molecule hybridizes with its complementary RNA molecule, leaving the non-template
DNA strand unpaired. These structures form normally during transcription initiation, IgG
class switching, and mitochondrial DNA replication. They also appear normally at telomeres1.
Abnormal R-Loop formation is associated with DNA damage, replication fork stalling, impaired
DNA repair, and genomic instability. Cancer cells with aberrantly activated transcriptional
programs can have transcription-replication conflicts, enhancing the likelihood of abnormal
R-loop formation.
CUT&Tag-IT R-loop Assay Kit Advantages:• Low background and high signal-to-noise ratio compared with other chromatin
profiling techniques• Faster and more efficient than RNA immunoprecipitation (RIP) and chromatin
immunoprecipitation (ChIP)• Compatible with 200,000 to 500,000 fresh or cryopreserved cells
This CUT&Tag-based approach to studying R-loops provides a powerful and highly sensitive
new tool for investigating the formation, regulation, and function of R-loops in various
biological contexts, ultimately advancing our understanding of this important
molecular process.
Product

FormatCatalog No.

CUT&Tag-IT™ R-loop Assay Kit

16 rxns

53167

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1

Kit Components and Storage
Please store each component at the temperature indicated in the table below.
This kit ships in two boxes so that components that can not freeze, such as SPRI Beads, do
not freeze in shipment, and the components that must stay frozen, such as the enzymes,
stay frozen. Be sure to store items at their correct storage temperature as indicated in thistable below.
All components are guaranteed for 6 months after receipt when stored properly.
Reagents

Quantity

Storage

5% Digitonin

600 µL-20°C

R-loop RNase A

45 µL

-20°C

1 x 500 µL
2 x 100 µL-20°C

CUT&Tag-IT™ Assembled pA-Tn5 Transposomes

16 µL

-20°C

10 µg/µL Proteinase K

80 µL

-20°C

DNA-RNA Hybrid mAb (Clone S9.6), 1 mg/mL

50 µL

-20°C

Rabbit Anti-Mouse Antibody

16 µL

-20°C

Tagmentation Buffer

1.5 mL4°C or -20ºC

Q5 Polymerase Master Mix

425 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

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

1X Binding Buffer

5.5 mL

4°C

Dig-Wash Buffer

57 mL

4°C

Concanavalin A Beads

320 µL

4°C

1 mL

4°C

55 mL

4°C

Protease Inhibitor Cocktail

SPRI Beads
Dig-300 Buffer

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2

Reagents

Quantity

Storage

Antibody Buffer

800 µL

4°C

1% IGEPAL

220 µLRT

0.5 M EDTA

250 µLRT

10% SDS450 µLRT

DNA Purification Wash Buffer*

10 mL

RT

DNA Purification Elution Buffer

5 mL

RT

DNA Purification Binding Buffer**

6 mL

RT

500 µLRT

16

RT

3 M Sodium Acetate
DNA Purification Columns SF*DNA Purification Wash Buffer must be reconstituted to a final concentration of 80% ethanol
prior to use. Add 40 mL of 100% ethanol to the DNA Purification Wash Buffer prior to use.
**DNA Purification Binding Buffer must be reconstituted to a final concentration of 60%
isopropyl alcohol prior to use. Add 9 mL of 100% isopropanol to the DNA Purification Binding
Buffer bottle prior to use.

Additional Materials Required• Ethanol, 80%• Ethanol, 100%
• Isopropanol, 100%
• 1X PBS• Microcentrifuge• Vortexer• Magnetic bar or magnetic plate rack for 0.2 mL tubes• 0.2 mL PCR tubes• 1.5 or 2 ml microcentrifuge tubes• Wet ice and ice bucket• Nutator or gentle orbital shaker, not an end-over-end rotator• Nutator or gentle orbital shaker that can be placed at 4°C, not an end-over-end rotator• 37°C water bath or incubator• Thermal cycler• Illumina® Sequencing System

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3

CUT&Tag-IT R-loop Assay Kit Protocol
Day 1 Binding Cells to Concanavalin A Beads (approximately 2 hours)
Prepare Buffers
Prepare Complete Antibody Buffer. Protease Inhibitor Cocktail must be added fresh.
Complete Antibody Buffer
IngredientNote:

1 Reaction

8 Reactions

16 Reactions

Antibody Buffer

50 µL

400 µL

800 µL

Protease Inhibitor Cocktail

0.5 µL

4 µL

8 µL

5% Digitonin

0.5 µL

4 µL

8 µL

1% IGEPAL

0.5 µL

4 µL

8 µL

This protocol is optimized for non-adherent cells. If adherent cells are to
be used, avoid using Trypsin or cell detachment solutions that digest cell
membrane proteins. Cell membrane proteins are necessary to bind the cells
to the Concanavalin A Beads in Section B of the assay. To lift adherent cells
from growth plates, use an enzyme-free dissociation method such as scraping
with a rubber policeman.

Prepare Cells (10 minutes)
Note:

When preparing beads, cells or nuclei, the buffers are kept on ice but the
samples are handled on the bench at room temperature for convenience.

1.

Resuspend the fresh or frozen cell pellet in 1X PBS for cell counting. Aliquot 200,000
to 500,000 cells per CUT&Tag-IT R-loop Assay Kit reaction. Cryopreserved cells canbe thawed on the bench or at 37°C water bath or heat block.

2.

Prepare Dig-Wash Buffer with Protease Inhibitor Cocktail. 2.1 mL of Dig-Wash Buffer
plus 21 µL Protease Inhibitor Cocktail are needed per sample.

3.

Centrifuge cells for 3 minutes at 600 x g at 4°C and remove the supernatant.

4.

Resuspend cells in 1 mL Dig-Wash (with Protease Inhibitor Cocktail).

5.

Centrifuge cells at 600 x g for 3 minutes at room temperature and remove
the supernatant.

6.

Repeat steps 4-5 for a total of two washes.

7.

Resuspend cells with 100 µL of Dig-Wash Buffer (with Protease Inhibitor Cocktail) per
each 200,000 to 500,000 cell sample.

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4Prepare Concanavalin A Beads (20 - 30 minutes)
8.

Resuspend the Concanavalin A Beads by vortexing. Aliquot 20 µL per sample into 0.2
mL PCR strip tubes. Resuspend beads as needed during aliquoting.

9.

Add 100 µL 1X Binding Buffer per sample.

10. Capture the Concanavalin A Beads on a magnetic plate, remove the supernatant, andresuspend the beads with 100 µL 1X Binding Buffer (mix by pipette).
11. Quickly spin the tubes down, capture the beads on a magnet, and remove
the supernatant.
12. Add 10 µL of 1X Binding Buffer to the beads.
13. Add the washed cells from step 7 to the Concanavalin A Beads, resuspend,
and incubate using a Nutator or orbital rotator at 20-30 RPM for 10 minutes atroom temperature.
DNA-RNA Hybrid (Clone S9.6) Antibody Binding (overnight)
14. Remove tubes from Nutator or orbital rotator and quickly centrifuge. Place the tubes
on a magnetic stand or bar to collect beads, and discard the supernatant.
15. Resuspend the beads/cells using 50 µL of ice-cold Complete Antibody Buffer by
gentle vortexing. Place tubes with samples on ice.
16. Add 5 µL molecular grade DNase/RNase-free water to samples, or 5 µL of RNase A
to negative control sample, and incubate at room temperature for 30 minutes on a
Nutator or orbital rotator at 20-30 RPM.
17. Add 2 µL of DNA-RNA Hybrid antibody to each sample with gentle pipetting.
18. Place samples on Nutator or orbital shaker overnight at 4°C at speed of 20-30 RPM.
Ensure caps are slightly elevated to avoid beads getting stuck in the lid. Liquid should
remain in the bottom and on the side of the tubes while rocking.

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5Day 2 (approximately 6 hours)
Bind Secondary Antibody
First, Prepare Buffers
Complete Dig-Wash Buffer
Ingredient

1 Reaction

8 Reactions

16 Reactions

Dig-Wash Buffer

1.3 mL

10.4 mL

20.8 mL

Protease Inhibitor Cocktail

13 µL

104 µL

208 µL

5% Digitonin

13 µL

104 µL

208 µL

1% IGEPAL

13 µL

104 µL

208 µL

1 Reaction

8 Reactions

16 Reactions

0.7 mL

5.6 mL11.2 mL

Protease Inhibitor Cocktail

7 µL

56 µL

112 µL

5% Digitonin

1.4 µL11.2 µL

22.4 µL

1 Reaction

8 Reactions

16 Reactions

100 µL

800 µL

1.6 mL

1 µL

8 µL

16 µL

0.2 µL1.6 µL3.2 µL

Complete Dig-300 Buffer
IngredientDig-300 Buffer

Complete Tagmentation Buffer
IngredientTagmentation Buffer
Protease Inhibitor Cocktail
5% Digitonin

Note:

Protease Inhibitor Cocktail must be added fresh.

20. Remove tubes from Nutator and give them a quick spin to collect samples in bottomof tubes. Then place tubes on magnetic stand or bar to pellet beads, and remove anddiscard the supernatant.
21. Wash the beads by adding 200 µL Complete Dig-Wash Buffer, resuspending thebeads, placing the tubes back on the magnetic stand or bar, and removing anddiscarding the supernatant.
22. Repeat step 21 two times for a total of 3 washes.
23. Dilute the Rabbit Anti-Mouse Antibody 1:100 in Complete Dig-Wash Buffer and add
100 µL per sample. Pipette gently to resuspend pellets.
24. Place the tubes on a Nutator at room temperature for 1 hour.

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625. Gently pipette samples to resuspend pellets, and place back on Nutator at room
temperature for an additional hour.
26. Remove tubes from Nutator and give the tubes a quick spin.
27. Place the tubes on a magnetic stand to pellet beads, and remove and discard
the supernatant.
28. Add 200 µL of Complete Dig-Wash Buffer. Gently pipette to resuspend the beads.
29. Repeat steps 27 - 28 twice for a total of 3 washes, removing and discarding thesupernatant after each wash.
Bind CUT&Tag-IT Assembled pA-Tn5 Transposomes (1 hour)
30. Mix CUT&Tag-IT Assembled pA-Tn5 Transposomes in Complete Dig-300 Buffer to a
final concentration of 1:100 for 100 µL per sample.
31. After a quick spin, place tubes on a magnetic stand to pellet beads, and remove anddiscard the supernatant.
32. Add 100 µL of the CUT&Tag-IT Assembled pA-Tn5 Transposomes in Complete Dig300 Buffer and gently pipette to resuspend the beads.
33. Place the tubes on a Nutator at 20-30 RPM at room temperature for an hour.
34. Remove tubes and quickly spin.
35. Place tubes on magnet or magnetic rack, and remove and discard supernatant.
36. Add 200 µL Complete Dig-300 Buffer to each sample and gently pipette toresuspend the beads.
37. Repeat steps 35 - 36 for a total of 3 washes.
Tagmentation (1 hour)
38. After a quick spin, place the tubes on a magnetic stand to pellet beads, and remove
and discard the supernatant.
39. Add 40 µL of Complete Tagmentation Buffer. Gently pipette to resuspend the beads.
40. Incubate at 37°C for 1 hour in a water bath or incubator.
DNA Extraction (1 hour)
41. To stop tagmentation and solubilize DNA fragments, add to each sample:
2.3 µL of 0.5 M EDTA
2.8 µL 10% SDS
0.5 µL 10µg/µL Proteinase K
42. Mix by vortexing at full speed for approximately 2 seconds and quick spin.
Note:

It is typical for the beads to form a large clump during this incubation.

43. Incubate the reaction for 1 hour at 55°C in thermal cycler with heated lid set to 65°C.

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744. Add 100 µL of water to each sample and mix with pipettor until bead/cell clump isthoroughly homogenized. Set pipettor to 100 µL to avoid pulling up air. Avoid making
bubbles as the SDS can cause the sample to become foamy. The samples will begooey, we suggest using a single channel pipettor and homogenizing one sample ata time.
45. After a quick spin, place the tubes on a magnet tray to clear and transfer supernatantto new microcentrifuge tubes.
Note:

Some of the beads will pull off with the DNA, this will not affect the
DNA purification.

46. Add 750 µl of DNA Purification Binding Buffer (with 60% isopropanol) to each sample
and mix by pipetting up and down. If the color indicator turns violet or orange add 8µL of 3 M Sodium Acetate.
47. For each sample, place a DNA purification column into a collection tube.
48. The following steps are performed at room temperature. Transfer each sample to its
corresponding column, close the cap, and centrifuge at 17,000 x g (approximately
14,000 rpm) for 1 minute.
49. Discard the flow-through and return the collection tube to the column.
Note:

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

50. Add 200 µL of DNA Purification Wash Buffer to the column and cap the column.
Centrifuge at 17,000 x g for 1 minute.
51. Discard the flow-through and repeat step 50 for a total of two washes.
52. Discard the flow-through and return the collection tube to the column. Centrifuge theempty tube at 17,000 x g for 2 minutes to remove any remaining DNA purification
Wash Buffer.
53. Transfer each column to a new microcentrifuge tube and add 22 µL of DNA
Purification Elution Buffer to the center of the column matrix, cap the column andincubate at room temperature for 1 minute.
54. Centrifuge at 17,000 x g for 1 min. Discard the column. DNA purification is complete.
The purified DNA can be stored at -20°C or you may proceed directly with the
PCR amplification.

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8

PCR Amplification (1 hour)
55. Set up the PCR reactions by adding the components in the order shown below, If
libraries are to be multiplexed for sequencing, ensure that a unique i5 - i7 index
combination is used for each sample.
Reagent

VolumeTagmented DNA Sample

20 µL

i5 Indexed Primer

2.5 µLi7 Indexed Primer

2.5 µLQ5 Polymerase Master Mix

25 µL

Total Volume

50 µL

56. Perform PCR using the following program on a thermal cycler (with a heated lid
to 105°C):
72°C for 5 minutes
98°C for 30 seconds
12 cycles of:
98°C for 10 seconds
63°C for 20 seconds
72°C for 1 minuteHold at 10°C
57. Perform a double-sided SPRI bead clean-up with 25 µL SPRI Bead Solution (0.5X
sample volume), pipette to mix and let stand 5 minutes.
58. Place on magnetic stand and move supernatant to new strip tube, and discard
the beads.
59. To the supernatant, add 35 µL SPRI Bead Solution (0.7X sample volume, bringing total
to 1.2X ratio). Let stand 5 minutes.
60. Place on magnetic stand and remove and discard supernatant. Wash beads twicewith 200 µL 80% Ethanol.
61. Allow beads to dry briefly, remove from magnetic stand when the beads turn fromshiny to matte in appearance, add 20 µL DNA Purification Elution Buffer, pipette tomix, and incubate for 1 minute at room temperature.
62. Place tubes on magnetic stand to pellet beads. Collect supernatant and transfer to a
new tube. Libraries are now ready for quantification and sequencing.

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9

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

TTCTGCCTTATCCTCT

N504

TATCCTCT

AGAGTAGA

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.

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10References
1.

Lin R., et al. (2021) Nucleic Acids Research Vol. 50, D303-D315

2.

Wang et al. (2021) Sci. Adv., 7:eabe3516

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11Troubleshooting Guide
Problem/Question

Recommendation

No visible nuclei pellet

The pellet may not be visible, proceed with protocol.
Be sure to position tubes with caps oriented in centrifuge so that you
know where the pellet should be in the tube, and proceed carefully
with next steps.

What is the recommended
sequencing depth?

25 million reads.

High backgroundBe sure to start with viable cells.
Check that cells/nuclei are intact by an automated cell counting
method such as the Countess II or by Trypan Blue staining anda hemocytometer.

Will the RNase A negative control
produce a library?

Yes. The RNase A treated sample will produce a library. Rnase A will
significantly deplete library concentration.

Should I use an IgG control?

No. We found IgG controls to be highly variable and inconsistent intagmentation-based assays. We recommend a no-DNA-RNA HybridmAb (Clone S9.6)antibody sample as a control.

Why does this kit use RNase A andnot RNase H like some publications?

We observed RNase H treatment did not completely abolish CUT&Tag
signals, and the extended overnight incubation of RNase H digestion
added more time to the protocol without a noticeable benefit.2

What percentage of mitochondrial
DNA reads are observed?

We observe a range of 36% to 67% of reads mapping to mitochondrial
reads. This will vary by cell type and cell treatment.

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12Technical Services

China

Japan

EuropeNorth America

If you need assistance at any time, please call or send an e-mail to Active Motif Technical
Service at one of the locations listed below.

Toll free:877 222 9543

Direct:

760 431 1263

Fax:

760 431 1351

E-mail:tech_service@activemotif.com

UK Free Phone:

0800/169 31 47

France Free Phone:

0800/90 99 79

Germany Free Phone:

0800/181 99 10

Direct:+32 (0)2 653 0001

Fax:

+32 (0)2 653 0050

E-mail:eurotech@activemotif.com

Direct:+81 (0)3 5225 3638

Fax:

+81 (0)3 5261 8733

E-mail:japantech@activemotif.com

Direct:(86)-21-20926090

Cell Phone:
E-mail:18521362870
techchina@activemotif.com

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13