CUT&Tag-IT™ Assay Kit,
Anti-Mouse Manual(version A4)
Catalog No. 53165 (16 rxns)
US Pat. No. 10,689,643, EP Pat. No. 2999784
and related patents and applications

Copyright 2021 Active Motif, Inc.

Revision

Date

Description of Change

A3

January, 2024

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

A4

April, 2024

Added notes to advise on optimalcell numbers

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 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© 2021 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 their respective
companies.Oligonucleotide sequences © 2019 Illumina, Inc. All rights reserved.
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
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purchaser, and are not to be resold. You agree not to reverse engineer or otherwise attempt todiscover the structure or composition of products or services deliverables unless we otherwise
agree in writing.

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TABLE OF CONTENTS

Page

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1
Kit Components and Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Kit Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Additional Materials Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Protocol
Section A. Prepare Fresh Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Section B. Bind Cells to Concanavalin A-Coated Beads. . . . . . . . . . . . . . . . . . . . . . . . . . 4
Section C. Bind Primary Antibody . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Section D. Bind Secondary Antibody. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Section E. Bind CUT&Tag-IT™ Assembled pA-Tn5 Transposomes . . . . . . . . . . . . . . . . . . 6
Section F. Tagmentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Section G. DNA Extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Section H. PCR Amplification. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Index Primers and Sample Sheet Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Troubleshooting Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Technical Services . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

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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 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 intheir 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 is optimized for 50,000 to 500,000 cells per reaction, although
200,000 cells are recommended. This Kit provides optimized reagents and protocol to produce
16 unique sequencing-ready Illumina®-compatible libraries.

CUT&Tag-IT™ Assay Kit Advantages• Compatible with as few as 5,000 cells• Low background signal enables lower sequencing depth• No artifacts caused by formaldehyde crosslinking

product
CUT&Tag-IT™ Assay Kit, Anti-Mouse

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formatcatalog no.

16 reactions

53165

Kit Components and Storage
The kit contains sufficient reagents for 16 CUT&Tag Assay reactions for use with a mouse primary
antibody to target or mark of interest. The reagents in this kit have multiple storage temperatures. Please store components according to the storage conditions below. All reagents areguaranteed stable for 6 months from date of receipt when stored properly.

CUT&Tag Assay Kit Reagents
Reagents

Quantity

Storage

5% Digitonin

610 µL-20°C

Concanavalin A Beads

320 µL

4°C

CUT&Tag-IT™ Assembled
pA-Tn5 Transposomes

16 µL

-20°C

Tagmentation Buffer

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

Rabbit Anti-Mouse Antibody

16 µL

-20°C

Protease Inhibitor Cocktail

1.42 mL-20°C

0.5M EDTA

68 µL

RT

10% SDS

20 µL

RT

10 µg/µL Proteinase K

18 µL

-20°C

16 columns

RT

DNA Purification Binding Buffer

10 mL

RT

DNA Purification Wash Buffer

12 mL

RT

DNA Purification Elution Buffer

560 µLRT

3M Sodium Acetate

128 µLRT

10 mM DNTPs

16 µL

-20°C

5X Q5 Buffer

160 µL-20°C

Q5 High Fidelity DNA Polymerase (2U/µL)

8 µL

-20°C

i5 Indexed Primer 1

10 µL

-20°C

DNA Purification Columns

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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• 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• Vortexer• Thermal cycler• Illumina® NextSeq® Sequencer• 1.5 mL Low-bind Microcentrifuge tubes• 2 mL Low-bind Microcentrifuge tubes• Microcentrifuge for 8-well PCR strip tubes with caps, or for 0.2 and 1.5 mL tubes• Multichannel pipette (20 - 200 µL)• Filter tips for pipettes

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CUT&Tag-IT™ Assay Kit, Anti-Mouse Protocol
This Kit is optimized for 50,000 to 500,000 cells, although 200,000 cells are recommended. This
Kit is for use with a mouse primary antibody to the histone mark of interest.
NOTE:

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 fromgrowth plates, use an enzyme-free dissociation method such as scraping with arubber policeman.

Section A. Prepare Fresh Cells (30 minutes)
NOTE:

Before using 1X Wash Buffer in Step 3 below, you must add Protease Inhibitor
Cocktail to the 1X Wash Buffer. Add 10 µL Protease Inhibitor Cocktail for every 1
mL of 1X Wash Buffer. 2.5 mL total are used per cell sample. Keep on ice. Wash
Buffer can be stored at 4°C for up to 1 week once Protease Inhibitor Cocktail has
been added.

1.

Harvest fresh culture(s) at room temperature and count cells. Place the desired amount ofcells into a 1.5 mL microcentrifuge tube.

2.

Centrifuge for three minutes at 600 X g at room temperature, then remove supernatant.

3.

Resuspend in 1 mL 1 X Wash Buffer at room temperature, then centrifuge for another threeminutes at 600 X g at room temperature, and remove supernatant.

4.

Resuspend in 1.5 mL 1 X Wash Buffer and transfer to a 2 mL microcentrifuge tube. Placetube with resuspended pellet on ice while the Concanavalin A beads are prepared in thenext steps.

Section B. Bind Cells to Concanavalin A Beads (30 minutes)
NOTE:

This Kit is optimized for 50,000 to 500,000 cells. Best results have beenobtained with 200,000 cells. 500,000 cells can become viscous in the steps
below (Bind Cells to Concanavalin A Beads, Steps 5-9) and DNA purification.

5.

Gently resuspend and withdraw enough of the Concanavalin A Bead slurry such that there
will be 20 µL per each sample. Use 20 µL for up to 500,000 cells. The following steps are
written for one sample of 500,000 cells. Do not adjust the volumes for lower cell numbers.

6.

Into a 2 mL 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 (30 seconds to 2 minutes).

7.

Withdraw the liquid completely and remove the tube from the magnetic stand. Add 1.5 mL 1
X Binding Buffer, mix by pipetting, and collect any liquid from the sides or cap with a quick
pulse spin in a microcentrifuge.
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8.

Place tube on magnetic stand to clear (30 seconds to 2 minutes), and remove liquid by
pipette. Resuspend in 20 µL 1 X Binding Buffer (20 µL per 500,000 cells per reaction) andkeep the tube containing prepared Concanavalin A Beads at room temperature until the
fresh cells are ready.

9.

Slowly add the bead slurry from Step 8 and invert tube to mix. Place the tube containing thecells and Beads on an end-over-end rotator for 10 minutes.

Section C. Bind Primary Antibody (2 hours to overnight)
NOTE:

Before using Antibody Buffer in Step 11 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.

10. 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), and remove the liquid via a pipette.
11.

Resuspend cells in a volume of ice-cold Antibody Buffer such that each sample is in a 50 µL
volume, not to exceed 500,000 cells per 50 µL. Gently vortex. Place the tube on ice.

12. Add 1 µL (or at least 1 µg) undiluted primary antibody to each sample, and gently vortex or
pipette to mix.
IMPORTANT: Use a mouse primary antibody. We recommend using a 1:50 to 1:100 dilution or the antibody manufacturer’s recommendation for the dilution used inapplications such as immunofluorescence. Histone H3K27me3 (Cat. No. 61018)
is a common mark that can be a positive control.
13. Incubate 2 hours to overnight at 4°C with orbital mixing, make certain liquid stays together
in the bottom of the tube so that it all mixes together.

Section D. Bind Secondary Antibody (60 minutes)
NOTE:

Before using Dig-Wash Buffer in Step 15 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.

14. 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.
15. Dilute the Rabbit Anti-Mouse secondary antibody 1:100 in Dig-Wash Buffer. 100 µL of thediluted secondary are needed per reaction. Add 100 µL of this diluted secondary antibody
to each sample while gently vortexing to dislodge the beads from the sides of the tube.
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516. Place the tubes on an orbital rotator at room temperature for 60 minutes.
17.

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.

18. Add 1 mL Dig-Wash Buffer to each reaction. Gently vortex or pipette to dislodge any beads
that have aggregated together.
19. Repeat Step 18 twice, for a total of three washes.

Section E. Bind CUT&Tag-IT™Assembled pA-Tn5 Transposomes(60 minutes)
NOTE:

Before using Dig-300 Buffer in Step 20 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.

20. 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.
21. 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.
22. Add 100 µL of the diluted CUT&Tag-IT™ Assembled pA-Tn5 Transposomes from Step 20 whilegently pipetting to make sure it mixes together.
23. Incubate the reactions at room temperature on an orbital rotator for 60 minutes.
24. 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.
25. Add 1 mL Dig-300 Buffer. Gently vortex or pipette to dislodge the beads.
26. Repeat Step 25 twice for a total of three washes.

Section F. Tagmentation (60 minutes)
NOTE:

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

27. 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.
28. Add 125 µL Tagmentation Buffer while gently vortexing or pipetting to mix each tube.
29. Incubate tubes at 37°C for 60 minutes.

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6Section G. DNA Extraction (60 minutes)
30. 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)
31. 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.

32. 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.
NOTE:

If the sample is too viscous, preventing the supernatant from being transferred,add 625 µL DNA Purification Binding Buffer, pipette up and down, place again
on the magnetic stand and transfer to a new tube. Proceed to step 34.

33. 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.
34. For each sample, place a labeled DNA Purification Column into a collection tube.
35. Transfer each sample to its corresponding column, close the cap, and centrifuge at 17,000 X
g (approximately 14,000 rpm) for 1 minute.
36. 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 thefirst use, to a final concentration of 80% (40 mL of 100% Ethanol to the DNA
Purification Wash Buffer bottle).

37. 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.
38. 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.
39. 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.
40. 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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7Section H. PCR Amplification
41.

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 (25 µM)

2.5 µLi5 Indexing Primer (25 µM)

2.5 µLdNTPs (10 mM)

1.0 µL5X Q5 Reaction Buffer

10 µL

Molecular grade nuclease-free water

3.5 µLQ5 Polymerase

0.5 µL
Total Volume = 50 µL

42. Perform PCR using the following program on a thermal cycler with a heated lid:
72°C for 5 minutes
98°C for 30 seconds
14 cycles of: 98°C for 10 seconds, 63°C for 10 seconds
72°C for 1 minuteHold at 10 °C

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843. 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 55 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 so that residual ethanol can evaporate. 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.

44. 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(NovaSeqv1.5 Reagent KitsiSeq,
MiniSeq, NextSeq,
HiSeq 3000/4000)

N501

TAGATCGCGCGATCTA

N502

CTCTCTATATAGAGAG

N503

TATCCTCTAGAGGATA

N504

AGAGTAGATCTACTCT

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

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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10References1.

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

Troubleshooting Guide
Problem/question

Recommendation

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 cells 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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11Technical Services
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.

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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