Enabling Epigenetics Research

ChIC/CUT&RUN Assay Kit
Catalog No. 53180(Version A6)

Copyright 2023 Active Motif, Inc.

Revision

Date

Description of Change

A2July, 2023

Updated Kit photo
Corrected typo on page 2

A3

October, 2023

Updated storage temperature range for 0.1 M CaCl2 and Updated
SPRI volume from 1.1 to 1.2 in step 3 page 9

A4

December, 2023

Corrected DNA Binding Buffer to DNA Purification Binding Buffer on
page 4

A5

May, 2024

Corrected SPRI bead/sample ratio from 1.5X to 1.6X in step 3 page 9A6July, 2024

Edited step 26 of ChIC/CUT&RUN DNA Isolation and Purification
Added NGS Library Prep option for Actie Motif’s DNA Library Prep
Kit for Illumina® (Cat. No. 53220)

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.
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
ChIC/CUT&RUN Assay Kit Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  4
Prepare Buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  4
Day 1 (approximately 3 hours) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  5
Day 2 (approximately 5 hours) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  7
NGS Library Preparation (3 hours) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  9
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Troubleshooting Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Technical Services . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

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Overview
CUT&RUN (Cleavage Under Targets & Release Using Nuclease) is an epigenetic method
used to investigate the genome-wide distribution of various chromatin-associated proteins
and their modifications1. CUT&RUN is similar to chromatin immunoprecipitation (ChIP), in that
it utilizes an antibody to target chromatin-associated marks and proteins, but requires lesssample material and less sequencing depths than ChIP1-3.
In CUT&RUN, a protein of interest is tagged with an antibody and bound to the chromatin
in intact cells. Then, a micrococcal nuclease (MNase) is used to cleave the DNA specificallyat the binding sites of the protein of interest. The MNase is fused to Protein A and Protein
G, and the Protein AG portion will attach to the antibody already bound to the protein ofinterest. Next, the MNase is activated by the addition of calcium, which in turn fragments
the DNA, allowing the DNA to enter the supernatant. The released fragments are purified,sequenced, and mapped to the reference genome to determine the protein’s binding sites.
The advantages of CUT&RUN over ChIP-Seq are a simplified protocol without sonication ofsamples, robust data from fewer cells (as few as 5,000 for histone modifications or 25,000
for some transcription factors) and shallow sequencing depth (as few as 2,000,000 reads forrobust marks or 25 reads on average for transcription factors)3.
CUT&RUN is a valuable tool for studying chromatin-associated proteins because it issensitive, specific, and requires fewer cells than ChIP, making it ideal for identifying
binding patterns of chromatin-associated proteins such as transcription factors or histone
modifications genome-wide. Chromatin-associated proteins play critical roles in regulating
various cellular processes such as gene expression, DNA replication, DNA repair, and celldifferentiation. Understanding the binding patterns of these proteins can provide insight into
how these cellular processes are regulated.

Product

FormatCatalog No.

ChIC/CUT&RUN Assay Kit

24 rxns

53180

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Kit Components and Storage
Please store each component at the temperature indicated in the table below. Kit andcomponents are guaranteed for 6 months after receipt when stored properly.
Reagents

Quantity

Storage

5% Digitonin

600 µL-20°C

RNase A (10 µg/µL)

40 µL

-20°C

2 x 500 µL-20°C

ChIC/CUT&RUN pAG-MNase

70 µL

-20°C

Glycogen 20 mg/mL

12 µL

-20°C

Histone H3K4me3 pAb, 10 µg, 1 mg/mL

8 µL

-20°C

Negative Control IgG, Rabbit

8 µL

-20°C

1 M Spermidine

28 µL

-20°C

Nuclei Isolation Buffer

2.7 mL

4°C

Concanavalin A Beads

300 µL

4°C

0.1 M CaCl2

45 µL

RT or 4°C

Stop Solution

1.1 mL

4°C

1X Binding Buffer

6 mL

4°C

Dig-Wash Buffer

55 mL

4°C

0.5 M EDTA

250 µLRT

DNA Purification Wash Buffer*

10 mL

RT

DNA Purification Elution Buffer

5 mL

RT

DNA Purification Binding Buffer**

6 mL

RT

500 µLRT

24

RT

7 strips

RT

Protease Inhibitor Cocktail, 500 µL

3M Sodium Acetate
DNA Purification Columns SF
0.2 mL Strip Tubes, Attached Caps

*DNA Purification Wash Buffer must be reconstitured 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 touse. Add 9 mL of 100% isopropanol to the DNA Purification Binding Buffer bottle prior to use.

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Additional Materials Required•

Antibody to protein of interest•

Ethanol, 100%•

Isopropanol, 100%•

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 orbital shaker•

Nutator or orbital shaker that can be placed at 4°C•

NGS Library Preparation kit compatible with small fragments such as
NEBNext® Ultra™ II DNA Library Kit for Illumina: E7645L•

Indexing primers such as NEBNext Multiplex Oligos for Illumina(Index Primers Set 1): E7335S•

Indexing primers such as NEBNext Multiplex Oligos for Illumina(Index Primers Set 2): E7500S•

DNA Library Prep Kit for Illumina® (Active Motif Cat. No. 53220)•

Dual Index Primers Set 1 for Illumina® (Active Motif Cat. No. 53221)•

Dual Index Primers Set 2 for Illumina® (Active Motif Cat. No. 53222)

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ChIC/CUT&RUN Assay Kit Protocol
Prepare Buffers
Prepare DNA Purification Binding Buffer: add 9 mL 100% isopropanol to the DNA Purification
Binding Buffer bottle to a final concentration of 60% isopropanol.
Prepare DNA Purification Wash Buffer: add 40 mL of 100% ethanol to the DNA Purification
Wash Buffer bottle, the final concentration of ethanol is 80%.
Prepare Stop Solution: Add 11 µL Glycogen to the vial of Stop Solution. Add 22 µL of RNase A
to Stop Solution. You will need 40 µL Stop Solution per reaction. Solution will be stable for 6
months at 4°C.
Before beginning, prepare Complete Wash Buffer, Cell Permeabilization Buffer and Antibody
Buffer. Some of each buffer will be the base component of the next buffer that follows insequence in the table below, and the remaining portions of each buffer will be stored asindicated in this table and used as indicated in the protocol.
Buffer Name

Components

1 Reaction 8 Reactions

24 Reactions

Complete Dig-Wash
Buffer

Dig-Wash Buffer

1.8 mL

14.4 mL

43.2 mL

Protease Inhibitor Cocktail

18 µL

144 µL432 µL1 M Spermidine

0.9 µL7.2 µL

21.6 µL

Cell Permeabilization Complete Dig-Wash Buffer
Buffer
5% Digitionin

1.4 mL11.2 mL

33.6 mL

2.8 µL

22.4 µL

67.2 µL

Antibody Buffer

Cell Permeabilization
Buffer

100 µL

800 µL

2.4 mL

0.5 M EDTA

0.4 µL3.2 µL

9.6 µL

Storage
4°C, for use
on Day 1

4°C, for use
on Day 2
Ice, for use
on Day 1

For nuclei isolation, (Step 13, optional but recommended) prepare Complete Nuclei Isolation
Buffer: You will need 100 µL per sample. Add 1.4 µL 1 M Spermidine to the Nuclei Isolation
Buffer bottle. When ready to use in the assay you will need to Add 10 µL 100X Protease
Inhibitior Cocktail (PIC) per 1 mL Nuclei Isolation Buffer.

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4Day 1 (approximately 3 hours)
Prepare Concanavalin A Beads (20 - 30 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 Concanavalin A Beads by vortexing.

2.

Dispense 10 µL resuspended Concanavalin A Beads per sample into 0.2 mL tubes
with attached caps. Beads should be resuspended periodically so the proper amountof bead slurry is pipetted into each sample if processing multiple samples at thesame time.

3.

Place tubes on magnetic plate rack or magnet bar.

4.

Remove supernatant from beads and discard.

5.

Add 100 µL 1X Binding Buffer per sample.

6.

Resuspend Concanavalin A Beads with 1X Binding Buffer by gently pipetting up
and down.

7.

Place tubes back on magnet to capture beads. Remove supernatant and discard.

8.

Repeat steps 5 - 7 for a total of 2 washes.

9.

Add 10 µL of Binding Buffer to each sample and store on ice until ready for
next steps.

Prepare Cells (10 minutes)
10. Prepare 5,000 to 500,000 cells, freshly grown, or thaw cryopreserved cells (5,000 to
500,000 per reaction). If you are starting with adherent cells, we recommend using
TrypLE Express for cell-dissociation. Cryopreserved cells can be thawed on the benchor at 37°C water bath or heat block.
11. Centrifuge cells at 600 x g for 3 minutes at room temperature.
12. Remove and discard supernatant.
Prepare Nuclei (optional but recommended) (15 minutes)
13. Prepare 100 µL per reaction of Complete Nuclei Isolation Buffer by adding 10 µL
Protease Inhibitor Cocktail and 0.5 µL 1 M Spermidine per 1 mL Nuclei Isolation Buffer
needed.
Note:

You may add 1.4 µL 1 M Spermidine to the full bottle of Nuclei Isolation
Buffer. The Nuclei Isolation Buffer with the added Spermidine can be stored
for 6 months at 4°C. Then, when ready to perform the assay, add 10 µL
fresh Protease Inhibitor Cocktail per 1 mL Complete Nuclei Isolation Buffer
per sample. You will need 100 µL of the Complete Nuclei Isolation Buffer
per sample.

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514. Gently resuspend cell pellets in 100 µL Complete Nuclei Isolation Buffer per up to
500,000 cells. This volume does not need to scale down for less than 500,000 cells.
Gently pipette or flick tube to ensure pellet is fully resuspended.
15. Place nuclei samples on ice for 10 minutes.
16. Centrifuge nuclei at 600 x g for 3 minutes at room temperature.
17. Remove and discard supernatant.
Wash Cells/Nuclei (30 minutes)
18. Gently resuspend cell or nuclei pellets in 100 µL Complete Dig-Wash Buffer per
sample (500,000 cells or nuclei per sample). Gently pipette or flick tube to ensure
pellet is fully resuspended.
19. Centrifuge cells or nuclei at 600 x g for 3 minutes at room temperature.
20. Remove and discard supernatant.
21. Repeat steps 18 - 20 for a total of 2 washes.
Note:

When cell number is limited or pellet is not easily visible the second wash canbe omitted.

22. Add 100 µL of Complete Dig-Wash Buffer per sample and gently resuspend by
pipetting or flicking tube until fully resuspended.
23. Transfer 100 µL of washed cells/nuclei to prepared Concanavalin A Beads.
24. Resuspend beads and cells/nuclei by gently pipetting up and down.
25. Allow to sit at room temperature for 10 minutes.
26. Place tubes on magnet or magnetic rack.
27. Remove and discard supernatant.
28. Add 50 µL of Antibody Buffer to each sample.
Antibody Binding (overnight)
29. Add 1 µg or 1 µL of appropriate antibody to each sample.
Note:

We recommend including samples for positive control Histone H3K4me3 andNegative Control IgG, Rabbit.

30. Resuspend by gently pipetting up and down.
31. 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.

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6Day 2 (approximately 5 hours)
Washes (10 minutes)
1.

Quick spin tubes and place tubes on magnet or magnetic rack at room temperature.

2.

Remove and discard supernatant.

3.

Add 200 µL Cell Permeabilization Buffer to each sample without disturbing pellet.

4.

Remove and discard supernatant.

5.

Repeat steps 3 - 4 for a total of 2 washes.

ChIC/CUT&RUN pAG-MNase Binding (20 minutes)
6.

Add 50 µL Cell Permeabilization Buffer to each sample.

7.

Add 2.5 µL of ChIC/CUT&RUN pAG-MNase to each sample.

8.

Remove tubes from magnet. Resuspend beads and mix by gently pipetting up
and down.

9.

Allow to sit at room temperature for 10 minutes.

10. Place tubes on magnet or magnetic rack.
11. Remove and discard supernatant.
12. Add 200 µL Cell Permeabilization Buffer to each sample without disturbing pellet.
13. Remove and discard supernatant.
14. Repeat steps 12 - 13 for a total of 2 washes.
15. Add 50 µL Cell Permeabiliaztion Buffer to each sample.
16. Place tubes on ice and pause for 2 minutes to allow samples to cool before
proceeding to the next steps.
Targeted Chromatin Digestion (3 hours)
17. Add 1 µL of 0.1 M CaCl2 to each sample.

18. Mix by gently pipetting up and down to resuspend beads and place back on ice.
19. Place on Nutator or orbital shaker at 4°C for 2 hours at speed of 20-30 RPM.
20. Remove from Nutator or orbital shaker and place samples on ice.
21. Add 40 µL of Stop Solution to each sample.
22. Mix samples by gently pipetting up and down.
23. Incubate samples at 37°C for 10 minutes in a thermal cycler. Set lid to 65°C. This step
releases the chromatin fragments into the supernatant.
24. Place tubes on magnet or magnetic rack for at least 30 seconds.

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ChIC/CUT&RUN DNA Isolation and Purification (45 minutes)
25. Carefully transfer samples to fresh 1.5 mL microcentrifuge tubes, avoiding any beads.
Note:

It is recommended to transfer the supernatant to a fresh tube and place onmagnet or magnetic rack to ensure no beads/cells are transferred in thenext steps.

26. Be sure that fresh isopropanol has been added to the bottle of DNA Purification
Binding Buffer to a final percentage of 60% to DNA Purification Binding Buffer before
this step. To each sample tube, add 450 µL of DNA Purification Binding Buffer. If thecolor of the DNA Purification Binding Buffer turns violet or orange, add 5 µL of 3 M
Sodium Acetate.
27. Vortex briefly and let stand 1 minute at room temperature.
28. Transfer sample and buffer to DNA Purification Columns SF.
29. Centrifuge at 16,000 x g for 1 minute.
30. Pour off flow through.
31. Add 200 µL DNA Purification Wash Buffer to each column.
32. Centrifuge columns at maximum speed (16,000 x g) for 1 minute.
33. Repeat steps 31 - 32 for a total of 2 washes.
34. Pour off flow through.
35. Centrifuge at maximum speed (16,000 x g) for 1 minute to ensure all traces of DNA
Purification Wash Buffer are removed.
36. Transfer column to new collection tube (collection tubes can be 1.5 mL
microcentrifuge tubes with the lids removed).
37. To each column, add 55 µL DNA Purification Elution Buffer.
38. Let stand at room temperature for 1 minute.
39. Centrifuge at maximum speed (16,000 x g) for 1 minute.
40. Transfer eluted ChIC/CUT&RUN DNA to new tubes. Store at -80°C if not being used
immediately for NGS Library Preparation. Samples can be stored for up to 1 week.
Your samples are now ready for NGS Library Preparation.

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NGS Library Prep (3 hours) Using Active Motif’s DNA Library Prep Kit for
Illumina® (Cat. No. 53220) (Optional)
1.

Follow the manual instructions for Active Motif’s DNA Library Prep Kit for Illumina®
(Cat. No. 53220), 39 µL of ChIC/CUT&RUN Assay Kit sample eluate are used per NGS
Library. You will also need Dual Index Primers Set 1 for Illumina® (Cat. No. 53221) orDual Index Primers Set 2 for Illumina® (Cat. No. 53222).

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NGS Library Prep (3 hours) Using NEBNext® Ultra™ II for DNA Library
Preparation for Illumina (Optional)
1.

These instructions were developed with the NEBNext® Ultra™ II for DNA Library
Preparation for Illumina. Follow the NEBNext® Ultra™ II for DNA Library Preparation forIllumina instructions with the additional steps below. You will also need the NEBNext
Multiplex Oligos for Illumina.

IMPORTANT!

Follow the NEBNext® Ultra™ II for DNA Library Preparation for Illumina
instructions. The steps below are additional tips.

2.

Dilute ligation adapter to 1:25 concentration (0.6 µM). Proceed to Step 2 of the
NEBNext® Ultra™ II for DNA Library Preparation instructions.

3.

For Step 3, SPRI bead cleanup, perform a double-sided cleanup with 0.4X bead/sample ratio followed by a 1.6X bead/sample ratio. For the first SPRI beans cleanup,
multiply sample volume x 0.4. For the second SPRI cleanup multiply sample volume x
1.2. Perform Steps 3.A1 through 3.A9. Do not perform Step 3B.

Note:

Measure sample volumes before SPRI cleanup as high viscosity can lead todeviation from expected volume.

4.

For Step 3A.10 perform a quick spin.

5.

For Step 3A.12 elute the beads in 17 µL of DNA Purification Elution Buffer.

6.

Follow Step 4 of the NEBNext® Ultra™ II for DNA Library Preparation instructions
and perform 12 PCR cylces for PCR enrichment of CUT&RUN libraries. Follow the
instructions for your Multiplex Oligos Primer Set.

7.

Following PCR enrichment, perform a double-sided SPRI bead cleanup on the PCR
product. Perform a double-sided cleanup with 0.4X bead/sample ratio followed by a
1.2X bead/sample ratio.
A. Add 20 µL SPRI beads for 50 µL PCR product (0.4X bead/original sample ratio)
and pipette up and down 10 times to mix.
B. Incubate at Room Temperature for 5 minutes.
C. Place on magnetic stand for 5 minutes to pellet.
D. Move supernatant to new tubes and discard beads from previous tubes.
E. Perform a second cleanup by adding 40 µL SPRI beads to the tubes with samples(1.2X bead/original sample ratio) and pipette up and down 10 times to mix.
F. Incubate for 5 minutes at Room Temperature and place on magnetic stand for 5
minutes to pellet beads.
G. Remove supernatant and discard.
H. Without removing the tubes from the magnetic stand, gently wash beads with
200 µL of 80% Ethanol.
I. Aspirate and discard supernatant.
J. Perform a second wash by repeating steps 7H - 7I.

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K. Using a P10 pipette, remove any leftover Ethanol from the tubes and let air dry
for approximately 5 minutes at Room Temperature, without over-drying (look forbeads going from shiny to matte).
L. Remove tubes from magnet and reconstitute beads with 22 µL of DNA Purification
Elution Buffer.
M. Incubate for 1 minute at Room Temperature.
N. Move tubes back to magnetic stand and allow 5 minutes to pellet beads at
Room Temperature.
O. Transfer 20 µL of libraries to fresh strip tubes. Store at -20°C or proceed toIllumina NGS sequencing.

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References
1.

Schmid, M. et al. (2004) Mol. Cell., 16(1): 147-157

2.

Skene, P.J. et al. (2017) Elife 6, e21856

3.

Skene, P.J. et al. (2018) Nat. Protoc., 13, 1006-1019

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12Troubleshooting Guide
Problem/Question

Recommendation

No visible nuclei pellet

The pellet may not be visible.
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 for transcription factors. For some histone marks you may
go down to 2 million reads.

How many cells should I
use for transcription factor
targets?

We recommend 500,000 cells and a CUT&RUN validated antibody.

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 and a hemocytometer.

Will the negative control
produce a library?

Yes. The negative control IgG will produce a library.

SPRI beads are not pelleting
towards the bottom or taking
too long to pellet.

Using a bar magnet, start by pelleting the SPRI beads towards the top of
the tube and gradually work your way down to the bottom of the tubes.
After, place the tubes on a magnetic plate.

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13

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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