Fixed Cell ATAC-Seq Kit Manual
Catalog No. 53151(version A2)

Copyright 2023 Active Motif, Inc.

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, inwhole 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.
© 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 their respective
companies.
For Research Use Only. Not for use in diagnostic procedures.

www.activemotif.com

TABLE OF CONTENTS

Page

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1
Kit Components and Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Reagents for Fixed Cell ATAC-Seq. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Additional Materials Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Fixed Cell ATAC-Seq Protocol. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Chromatin Opening and Cell Permeabilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Tagmentation Reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Releasing and Decrosslinking Tagmented DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
DNA Purification of Tagmented DNA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
PCR Amplification of Tagmented DNA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Index Primers and Sample Sheet Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Recommended Cell Fixation Protocol. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Troubleshooting Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Technical Services . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .14

www.activemotif.com

Overview
The Assay for Transposase-Accessible Chromatin via Sequencing (ATAC-Seq) method was first
introduced in 20131. ATAC-Seq is a rapid assay of the epigenetic state by enabling identificationof open chromatin regions. In the assay, intact nuclei are treated with a hyperactive Tn5 transposase mutant which is able to simultaneously tag the target DNA with sequencing adapters andfragment the DNA in a process termed “tagmentation”2.
Because of the assay’s speed, simplicity, sensitivity, and applicability to a wide range of sample
types, ATAC-Seq has become a commonly used epigentic assay, and can serve as a gateway tofurther, more detailed, epigenetic analyses.
The Fixed Cell ATAC-Seq Kit provides the reagents necessary to produce 16 unique sequencingready Illumina®-compatible ATAC-Seq libraries from 50,000 - 100,000 formaldehyde-fixed cells
per reaction.

Fixed Cell ATAC-Seq Advantages• Assess the epigenetic profile of open chromatin regions in formaldehyde-fixed cell samples• Yields next-gen sequencing-ready processed samples in 2 days
• Simple and rapid four-step protocol

product
Fixed Cell ATAC-Seq Kit
Note:formatcatalog no.

16 reactions

53151

The Fixed Cell ATAC-seq Kit is for research use only. Not for use in diagnostic procedures.
Illumina® is a registered trademark of Illumina, Inc.

www.activemotif.com

1

Kit Components and Storage
The kit contains sufficient reagents to produce 16 unique next-gen sequencing-ready ATAC-Seq
libraries. The reagents in this kit have multiple storage temperatures. The Fixed Cell ATAC-Seq
Kit is shipped at two temperatures, with one box on dry ice for components to be stored at -20°C,
and a second box at room temperature for components to be stored at 4°C. Please store components according to the storage conditions below. All reagents are guaranteed stable for 6 months
from date of receipt when stored properly.
Reagents

Quantity

Storage

Hypotonic Buffer

1.7 mLRT

Hypotonic Quench

50 µL

4°C

Releasing Buffer

1.3 mlRT

Proteinase K (10 µg/µl)

70 µl

-20°C

RNase A (10 µg/µl)

40 µl

-20°C

Protease Inhibitor Cocktail (PIC)

22 µL

-20°C

Assembled Transposomes

205 µl-20°C

2X Tagmentation Buffer

425 µl-20°C

10X PBS500 µl4°C

10% Tween 20

10 µl

RT

1.0% Digitonin

10 µl

-20°C

0.5 M EDTA

250 µLRT

16 columns

RT

DNA Purification Binding Buffer

5.5 mlRT

DNA Purification Wash Buffer

10 ml

RT

DNA Purification Elution Buffer

5 ml

RT

3 M Sodium Acetate

450 µlRT

dNTP mix, 10 mM

40 µl

-20°C

2 x 130 µl

-20°C

10 µl

-20°C

DNA Purification Columns SF

5X Q5 Buffer
Q5 High-Fidelity DNA Polymerase (2U/µl)

www.activemotif.com

2

Reagents

Quantity

Storagei7 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

SPRI Beads

1 ml

4°C

Additional Materials Required• 100% Ethanol• Deionized water• Trypan Blue
• Pipettes and corresponding pipette tips
• Refrigerated Microcentrifuge• Swing-bucket centrifuge• Low-bind 1.5 ml microcentrifuge tubes• 300 µl PCR tubes (PCR strip tubes are recommended)• Bar magnet or magnetic plate• 37°C incubator• Microcentrifuge tube orbital rotator that can be place in 37°C incubator• A dry block heater can be used instead of the 37°C incubator• Thermal Cycler with heated lid• Thermal Shaker or Thermomixer• Ice and ice bucket

www.activemotif.com

3

Fixed Cell ATAC-Seq Kit Protocol
Note: We recommend fixing cells in fresh formaldeyde. Please see the Appendix at theend of this manual for details. Cells that were fixed in not freshly prepared formaldehyde work in the assay but yield less robust sequencing results.

Chromatin Opening and Cell Permeabilization - Day 1
NOTE: This protocol is written for one single reaction of 50,000 cells per reaction. We
recommend 50,000 to 100,000 cells per reaction.
You will need to add 1 µL Proteinase Inhibitor Cocktail to 99 µL Hypotonic Buffer
per reaction, prepared fresh.
Place Hypotonic Quench on ice for step 7. You will need 2 µL per sample.
1.

Pre-heat thermal shaker with microcentrifuge tube adapter to 62°C.

2.

Prior to retrieving cells, add 1 µL of Proteinase Inhibitor Cocktail to 99 µL room temperature
Hypotonic Buffer (per sample).

3.

Put your fixed cell samples on ice and do not thaw. We recommend 50,000 cells per
sample.

4.

Resuspend each cell pellet in 100 µL room temperature Hypotonic Buffer by gently pipetting
in a 1.5 mL microcentrifuge tube.

5.

Place the microcentrifuge tube containing the cells into the preheated thermal shaker, set
the RPM to 600, press start, and incubate for 10 minutes.

6.

During the 10 minute incubation, label 1 PCR tube per sample, and keep on ice.

7.

After 10 minutes, remove the microcentrifuge tube containing the cells from the thermal
shaker and add 2 µL of cold Hypotonic Quench directly to the tube. At this stage you now
have processed lysed cells.

8.

Place each microcentrifuge tube containing the cells in the orbital rotator and place insideinside the 37°C incubator. Set the orbital rotator to 10 RPM and incubate for 15 minutes.
NOTE: As an alternative, the microcentrifuge tubes can be placed on a dry heat block set
to 37°C, while gently flicking the tubes every 10 minutes.
www.activemotif.com

4

9.

During the 15 minute incubation, make the Tagmentation Reaction Buffer and keep on ice.
NOTE: The Assembled Transposomes will be added to the Tagmentation Reaction Buffer
at a later step. Keep the Assembled Transposomes at -20°C until it is required. In
step 12 below you will add 12 µL of Assembled Transposomes to this Tagmentation
Reaction Buffer.
Tagmentation Reaction Buffer

Per Sample

Reagent

Volume2X Tagmentation Buffer

25 µL

10X PBS

2 µL

1% Digitonin

0.5 µL

10% Tween 20

0.5 µL

Total Volume

28 µL

10. Retrieve the sample tubes from the incubator and centrifuge at 1000 x g for 5 minutes at
4°C.
11.

Carefully remove supernatant, leaving behind 15 µL, ensuring the cell pellet is not disturbed. Discard supernatant. Leave some supernatant, approximately 15 µL in the tube
containing the cell pellet.

12. Add 12 µL of Assembled Transposomes to the ice-cold Tagmentation Reaction Buffer fromstep 9. Mix by pipetting up and down.

Tagmentation Reaction - Day 1
13. Add 40 µL of Tagmentation Reaction Buffer containing the Assembled Transposomes toeach sample. Gently pipette to resuspend cells in the Tagmentation Reaction Buffer. Keepon ice.
14. Transfer 50 µL of the resuspended pellet in the Tagmentation Reaction Buffer to the corresponding pre-labelled and pre-chilled PCR tube from step 6. Quickly pulse spin the PCR
tubes in mini-centrifuge to collect all liquid together in the bottom of the tubes.
15. Incubate the PCR tubes at 37°C with no shaking for 60 minutes.
16. After the incubation, quickly spin the PCR tubes with a single pulse on a mini-centrifuge
to collect all liquid together in the bottom of the tubes and add 4.2 µL of 0.5 M EDTA per
sample to the PCR tubes. Gently vortex the PCR tubes for 1 second at low speed and set the
tubes at room temperature for 15 minutes.
17.

During the incubation, label a low-bind microcentrifuge tube, corresponding to the PCR
tubes.

18. After the incubation, transfer all of the tagmented DNA to the corresponding microcentrifuge tube labeled in the previous step.
www.activemotif.com

5Releasing and Decrosslinking Tagmented DNA - Day 1
19. Centrifuge the microcentrifuge tubes at 12,000 x g for 3 minutes at 4°C.
TIP:

If the microcentrifuge tubes are all oriented in the centrifuge with their hinges
facing outward, the pellet will be on the hinge side of the tube. At this step, thetagmented DNA is in the pellet, this is the ATAC-Seq reaction sample.

20. The protocol steps continue at room temperature after the centrifugation in Step 19. Carefully remove and discard 42 µL of the supernatant. The pellet will be hard to see. 10 µL
should be left behind, be very careful not to disturb the pellet.
21. Add 75 µL of room temperature Releasing Buffer to the pellet, and resuspend by gently
pipetting 20 to 30 times. At this point, you are releasing the tagmented DNA from the pellet.
22. Give the sample a quick vortex at low setting. Then, incubate the resuspended pellet atroom temperature for 30 minutes. Every 10 minutes during this 30 minute incubation, gently
vortex on low setting for 1 second. At this point there is no active protein, and the DNA isreleased from the pellet.
23. Centrifuge the tubes at 17,500 x g for 15 minutes at 4°C. This is removing the large molecular weight fragments.
During this step, thaw the Proteinase K and RNaseA at room temperature. Once thawed,
place on ice. Label PCR tubes for each sample for the next steps.
24. Retrieve sample tubes from the centrifuge and transfer 56 µL of the supernatant to the corresponding new PCR tube.
25. Add 2 µL Proteinase K and 2 µL RNase A directly to each sample.
26. Gently vortex the PCR tubes for 1 second at low speed and quickly spin the PCR tubes with asingle pulse in a mini-centrifuge to collect all liquid at the bottom of the tubes.
27. Put PCR tubes into a thermal cycler and run the following overnight decrosslinking programwith the volume set at 60 µL and the lid temperature set to 105°C:
Decrosslinking Program
37°C for 30 minutes
55°C for 4 hours
65°C for 12 hours
65°C hold
NOTE: The 65°C hold is so you can continue the assay the following day. This is the endof Day 1. The DNA Purification must be done on the following day, not more than 18hours later.

www.activemotif.com

6

DNA Purification - Day 2 (3 - 4 hours)
28. Label a low-bind 1.5 mL tube to correspond with the sample.
29. Add 300 µL of room temperature DNA Purification Binding Buffer to each labeled microcentrifuge tube.
30. Add 5 µL Sodium Acetate directly to each microcentrifuge tube containing the DNA Purification Binding Buffer.
31. Retrieve the PCR tubes containing the decrosslinked and tagmented DNA from the thermal
cycler. Let the tubes sit at room temperature for 1 minute before proceeding to the nextstep.
32. Gently spin the tubes with a single pulse on a mini-centrifuge to collect all liquid at the bottom of the tubes.
33. Trasfer all 60 µL of the sample from the PCR tube to the corresponding 1.5 mL tube containing the DNA Purification Binding Buffer. The color should be yellow. If the color is anythingother than yellow, add 3 M Sodium Acetate in 5 µL increments until the color is yellow.
Pipette up and down to mix the sample.
a. For each sample, place a labeled DNA Purification Column into a collection tube.
b. Transfer each sample to its corresponding column, close the cap, and centrifuge at
17,000 x g for 1 minute.
c. 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 a new bottle of
DNA Purification Wash Buffer).
d.

Add 750 µL of DNA Purification Wash Buffer to the column and cap the column.
Centrfuge at 17,000 x g for 1 minute.
e. Discard the flow-through and the collection tube. Transfer the column to an empty
microcentrifuge tube. Centrifuge the empty tube with column at 17,000 x g for 2 minutes toremove ay remaining DNA Purification Wash buffer. Discard the microcentrifuge tube after
spinning.
f.
Transfer each column to a new microcentrifuge tube and add 35 µL DNA Purification
Elution Buffer to the center of the column matrix. Cap the column and incubate at room
temperature for 1 minute.
g. Centrifuge at 17,000 x g for 1 minute. Discard the column. DNA purification is complete.
This is now the purified tagmented DNA. The purified DNA can be stored at -20°C for up to 3
days or you may proceed directly to the PCR amplification.

www.activemotif.com

7

PCR Amplification of Tagmented DNA
Note: If a kit such as the KAPA Real-Time Library Amplification Kit is used, an initial 72°C
extension step is essential.
1.

Set up the PCR reactions by adding the components in the order shown below. If libraries
are to be multiplexed for sequencing on the same flow cell, ensure that a unique i5 and/or
i7 index combination is used 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:

2.

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

VolumeTagmented DNA

33.5 µl

i7 Indexed Primer (25 µM)

2.5 µl

i5 Indexed Primer (25 µM)

2.5 µldNTPs (10 mM)

1 µl

5X Q5 Reaction Buffer

10 µl

Q5 Polymerase (2 U/µl)

0.5 µlPerform PCR using the following program on a thermal cycler (with a heated lid):
72°C 5 minutes
98°C for 30 seconds
7 to 10 cycles of: 98°C for 10 seconds, 63°C for 30 seconds, 72°C for 1 minuteHold at 10°C.
During the PCR cycles, take SPRI beads out of 4°C and warm to room temperature.

www.activemotif.com

8

3.

Once the PCR reaction is complete, fully resuspend the SPRI beads by gently vortexing on
low setting for 1-2 seconds. Use the SPRI beads immediately after resuspension to avoid
beads settling at the bottom of the tube.
Perform double-sided SPRI clean-up with 25 µl SPRI bead solution for right side clean up
and 25 µL for left side clean up as follows:4.

a.

Prepare 400 µL of 80% ethanol per sample from 100% ethanol and nuclease freewater.

b.

Uncap the PCR tubes and add 25 µL of resuspended room temperature SPRI beads toeach tube containing the PCR product.

c.

Vortex the tubes briefly to mix.

d.

Incubate the samples at room temperature for 2 minutes.

e.

Apply magnet to sample tubes and wait 5 minutes for beads pellets to form.

f.

Keepping the tubes on the magnet, carefully uncap the tubes and transfer the supernatant to new PCR tubes, taking care to avoid the bead pellets. The tubes with thebeads may be discarded.

g.

Vortex the SPRI beads to resuspend. Add 25 µL of SPRI beads to each tube containing
the supernatant.

h.

Vortex the tubes briefly to mix.

i.

Incubate the samples at room temperature for 5 minutes.

j.

Apply magnet to sample tubes and wait 5 minutes for bead pellets to form. Remove
and discard supernatant.

k.

Keeping the tubes on the magnet and with the magnetic beads still pelleted, add
180 µL fresh 80% ethanol to each well, taking care not to disturb the pelleted beads.
Incubate for 30 seconds, aspirate, and repeat with another 180 µL of 80% ethanol.

l.

Aspirate the supernatant completely and air dry the beads until the beads are nolonger shiny (1 - 3 minutes).

m.

Remove tubes from the magnet and elute DNA by adding in 20 µL of DNA Purification
Elution Buffer to each tube.

n.

Vortex tubes briefly to mix and incubate for 5 minutes for bead pellet to form.

o.

Place the PCR tubes on the magnet, carefully remove eluate and transfer to fresh,labelled PCR tubes.

At this stage, libraries are ready for quantification and sequencing. Use a library quantification kit for next-generation sequencing to quantify the library (e.g. Roche Kapa Biosystems,
Catalog No. KR0405). PCR amplified libraries can also be analyzed to assess size distribution with a Bioanalyzer, TapeStation, or similar instrument to assess size distribution.

www.activemotif.com

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

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.

References
1.
2.
3.
4.

Buenrostro, J. D., et al. (2013) Nat. Methods 10: 1213-1218.
Adley, A., et al. (2010) Genome Biol. 11.
Corces, M. R. et al. (2017) Nat. Methods 14: 959-962.
Zhang, H., et al. (2022) BMC Genomics 23: 214.
www.activemotif.com

10

Recommended Cell Fixation Protocol
Note: The protocol below is designed to produce aliquots of 50,000 formaldehyde-fixed
cells per reaction.
Best results are obtained when fresh, methanol-free formaldehyde is used. We recommend 16%
formaldehyde that is provided in an ampule such as Pierce™ 16% Formaldehyde (w/v), Methanolfree Catalog Number 28906 available from Thermo Fisher Scientific. If preparing your own
Formalehyde Solution, the formaldehyde must be added fresh, just before using the solution.
PREPARE:

Cell Fixation Buffer

CompoundInitial Concentration

Final Concentration

Formaldehyde Solution(formaldehyde must be added
right before using if preparing
your own)

16%

11%

NaCl

5M

100 mM

EDTA, pH 8.0

250 mM

1 mM

HEPES, pH 7.5

1M

50 mM

Quantity Sufficient

Quantity Sufficient

Water

1.

Bring the cell suspension concentration to 1 - 2 x 106 cells/mL with complete cell culture
medium in a 15 mL tube (<10 mL of suspension) or 50 mL tube (10 - 30 mL of cells). If <1 x 106
cells, use 0.5 mL of the medium in a 1.5 mL tube.

2.

Gently vortex the cell suspension, add 1:10 (vol:vol) 10X Cell Fixation Buffer drop by dropwith a micropippetter and rotate at a low speed for 10 min. at room temperature.

3.

Stop the reaction by gently vortexing, and add 2.5 M glycine in the ratio of 1:20 (vol:vol).
Invert the tubes a few times and incubate on ice for 5 min.

4.

Perform the remaining steps at 4°C or on ice. Spin the tubes at 800 x g for 5 min at 4°C anddiscard the supernatant.

5.

Resuspend the pellet gently with 5 mL of ice-cold 1X PBS and incubate for 2 min. on ice.

6.

Repeat steps 4 and 5 with a volume of 1 mL of ice-cold 1X PBS that will bring the cell density
to 1 x 106 cells/mL. Aliquot 50,000 cells into precooled 1.5 mL tubes labeled for long-term
storage.
NOTE: We recommend aliquoting cells into 50,000 - 100,000 cell pellets. This eliminates
the need to count and aliquot stored cells after they are thawed. Cell pellets arethen snap frozen, and can be thawed directly in Hypotonic Buffer at 62°C for theassay.

7.

Spin the tubes at 1,200 x g at 4°C and remove as much of the supernatant as possible without disturbing the cell pellet. Snap freeze the tubes in liquid nitrogen, and store at -80°C.

www.activemotif.com

11Troubleshooting Guide
Question

AnswerWhy did my reaction
fail?

Pipetting 10 - 30 times and vortexing every 10 minutes in the Tagmentated DNA
Releasing step 22 is essential. The experiment will not work without this.

Why is there highbackground in thesequencing data?

Yes, high background is expected with fixed cells in ATAC-Seq. The backgroundis always higher in fixed cells versus native cells. Please see reference 4, Zhang
et al 2022.

Why was no library
produced?

Sample loss is very likely. Be sure to leave behind 15 µL of sample at removal of
Hypotonic Buffer stage (step 11) and after the high speed spin/buffer exchange
following tagmentation into Releasing Buffer (step 19).

Can all the reagentsand kit components,
including master mixes
and enzymes, thaw
out and be prepared atroom temperature?

Everything except the Assembled Transposomes and Q5 DNA polymerase canbe thawed at room temperature. The Assembled Transposomes and Q5 DNA
Polymerase are formulated with glycerol and will not need thawing, just put
these on ice. The other components can be frozen again and thawed again for
future reactions, so not all 16 reactions need to be done simultaneously.

Does sonicating fixedsamples generate better results?

No. We did not observe improvement in data generated with the assay bysonicating fixed cell samples.

What is the expected
final library size, andwhat should the library
traces look like on a
fragment analyzer?

Library fragments should range from around 250 bp to 1000 bp in length withan oscillation period ~150 bp. To see example traces, please visit the kit web
page at activemotif.com/atac-seq-kit

How many sequencing
reads are needed per
sample?

Typically 30 million paired-end reads is sufficient, with 20 million being a minimum. The number of reads is dependent both on the sample genome and theend goal of the ATAC-Seq assay. If the sample genome is very large, or if moreadvanced analysis is required, more sequencing reads may be needed.

Can I multiplex morethan 16 samples using
the ATAC-Seq Kit?

The Fixed Cell ATAC-Seq 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 liketo multiplex more than 16 samples our Nextera™-Compatible Multiplex Primers(96 plex) kit (Cat. No. 53155) enables multiplexing up to 96 reactions. These
primers are provided at a concentration of 25 µM to be used directly in our Kits.
You could also purchase and combine other Illumina Nextera primers at thesame concentration (25 µM) as those in the kit.

www.activemotif.com

12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

www.activemotif.com

13