Enabling Epigenetics Research

ATAC-Seq Kit
Catalog No. 53150(Version B12)

Copyright 2020 Active Motif, Inc.

Revision

Date

Description of Change

B11

February, 2024

Removed note from page 5.

B12July, 2024

Fixed error on page 6, step 3.

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.
© 2020 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.
Illumina® is a registered trademark of Illumina, Inc.

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Contents
Overview  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Kit Components and Storage  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  2
Additional materials required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  3
ATAC-Seq Kit Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  4
Tissue Sample Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  4
Cell Sample Preparation  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  6
Tagmentation Reaction and Purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  7
PCR Amplification of Tagmented DNA  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  8
Index Primers and Sample Sheet Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Troubleshooting Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Frequently Asked Questions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Technical Services . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

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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
identification of open chromatin regions. In the assay, intact nuclei are treated with ahyperactive Tn5 transposase mutant which is able to simultaneously tag the target DNA withsequencing adapters and fragment the DNA in a process termed “tagmentation”2.
Because of the assay’s speed, simplicity, sensitivity, and applicability to a wide range ofsample types, ATAC-Seq has become a commonly used epigenetic assay, and can serve
as a gateway to further, more detailed, epigenetic analyses. The ATAC-Seq Kit provides thereagents necessary to produce 16 unique sequencing-ready Illumina®-compatible ATAC-Seq
libraries from 20 - 30 mg tissue or 50,000 - 100,000 cells per reaction.
ATAC-Seq Advantages•

Assess the epigenetic profile of open chromatin regions•

Yields next-gen sequencing-ready processed samples in hours•

Simple and rapid three-step protocol
Product

FormatCatalog No.

ATAC-Seq Kit

16 rxns

53150

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Kit Components and Storage
The kit contains sufficient reagents to produce 16 unique next-gen sequencing-ready ATACSeq libraries. The reagents in this kit have multiple storage temperatures. The 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. Pleasestore components according to the storage conditions below. All reagents are guaranteedstable for 6 months from date of receipt when stored properly.
Reagents

Quantity

Storage

ATAC Lysis Buffer

17 mL

RT

Assembled Transposomes

170 µL-20°C

2X Tagmentation Buffer

425 µL-20°C

1X PBS2 x 1 mL

-20°C or RT

10X PBS500 µLRT

10% Tween 20

10 µL

RT

1.0% Digitonin

10 µL

-20°C

16 columns

RT

DNA Purification Binding Buffer

4.5 mLRT

DNA Purification Wash Buffer

10 mL

RT

DNA Purification Elution Buffer

5 mL

RT

3 M Sodium Acetate

450 µLRT

10 mM dNTPs

40 µL

-20°C

5X Q5 Buffer

2 x 130 µL-20°C

Q5 High-Fidelity DNA Polymerase (2U/µL)

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

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

DNA Purification Columns

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Additional Materials Required•

100% Ethanol•

Deionized water•

Trypan Blue•

Pipettes and corresponding pipette tips•

Microcentrifuge•

Swing-bucket centrifuge•

1.5 mL microcentrifuge tubes•

300 µL PCR tubes•

Bar magnet•

40 µm filter (for tissue samples)•

Razor blade (for tissue samples)•

5 cm petri dish (for tissue samples)•

15 mL conical tubes (for tissue samples)•

Ice-cold PBS (for tissue samples)•

Dounce homogenizer (for tissue samples)

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ATAC-Seq Kit Protocol
Note:

The protocols below are designed for 20 to 30 mg tissue or 50,000
to 100,000 cells.

Tissue Sample Preparation
This protocol is designed for 20 to 30 mg of tissue per reaction. Fresh tissue can be used or
tissue that has been flash frozen at -80°C.
Take SPRI beads out of 4°C and warm to room temperature by simply placing the vial of SPRI
beads on the bench top.
1.

For each sample, label a 5 cm petri dish and place on wet ice, along with a labeled 15
mL conical tube containing 5 mL ice-cold PBS.

2.

Transfer each sample to its corresponding dish, mince with a razor blade, and transfer
to the corresponding 15 mL conical tube containing ice-cold PBS using a 1 mL pipettetip (the tip can be cut to widen the bore in order to avoid clogging the tip).

3.

Centrifuge the 15 mL conical tubes at 500 x g for 5 minutes at 4°C.

4.

Aspirate PBS from centrifuged tube, and add 1 mL ATAC Lysis Buffer.

5.

Using a 1 mL pipette tip with a widened bore, transfer each sample to a 1 mL dounce
homogenizer and dounce slowly for 30 strokes using a tight-fitting pestle (forexample, small-clearance, type B).

6.

Filter each homogenized sample through a 40 µm mesh strainer and collect in a
fresh 1.5 mL microcentrifuge tube. Immediately after filtration, take a 10 µL aliquot forcell counting.

7.

Count the cells in each aliquot using Trypan Blue cell-viability staining (stain in a 1:1
ratio of cell volume:0.4% Trypan Blue). Only nuclei stained blue by Trypan Blue should
be counted.

8.

Invert cell samples gently to mix, then aliquot 50,000 - 100,000 cells into a new tube.

9.

Centrifuge the new cell aliquots at 500 x g at 4°C for 5 minutes. During this time,
prepare the Tagmentation Master Mix.
Tagmentation Master Mix (per sample)
Reagents

Volume2X Tagmentation Buffer

25 µL

10X PBS

2 µL

1.0% Digitonin

0.5 µL

10% Tween 20

0.5 µL

H 2O

12 µL

Assembled Transposomes

10 µL

10. Aspirate supernatant and proceed immediately to the Tagmentation Reaction and
Purification steps.

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4Cell Sample Preparation
50,000 to 100,000 fresh cells or cryopreserved frozen cells can be used. Cells must be ofhigh quality to preserve viability. Cryopreserved cells should be cryopreserved in a controlledrate freeze with media formulated to protect against the ice crystal formation and subsequentcell damage.
Take SPRI beads out of 4°C and warm to room temperature by simply placing the vial of SPRI
beads on the bench top.
1.

Count cells and aliquot 50,000 to 100,000 cells into a fresh 1.5 mL centrifuge tube foreach sample.

2.

Centrifuge the cells at 500 x g for 5 minute at 4°C. Orient the tubes such that thepellet will be where you expect it to be. If there is o visible pellet, spin an additional 5
minutes at 1,000 x g at 4°C.

3.

Gently remove supernatant by pipetting and add 100 µL of ice-cold PBS. Do notresuspend or disturb pellet. Spin once more at 500 x g for 5 minutes at 4°C.

4.

Remove supernatant very carefully, ensuring to not disturb the cell pellet. By
pipetting, thoroughly resuspend the cell pellet in 100 µL ice-cold ATAC Lysis Buffer.

5.

Transfer the resuspended cell pellet to a PCR tube on ice. Immediately spin down at
500 x g for 10 minutes at 4°C. During this time, prepare the Tagmentation Master Mix.
Tagmentation Master Mix (per sample)
Reagents

6.

Volume2X Tagmentation Buffer

25 µL

10X PBS

2 µL

1.0% Digitonin

0.5 µL

10% Tween 20

0.5 µL

H 2O

12 µL

Assembled Transposomes

10 µL

After the spin, remove the supernatant very carefully, ensuring the cell pellet
is not disturbed (this is a critical step; take extra care to remove the ATAC Lysis
Buffer but not lose nuclei). Immediately continue to the Tagmentation Reaction and
Purification steps.

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Tagmentation Reaction and Purification1.

Add 50 µL of Tagmentation Master Mix to each sample (does not have to be onice). Gently pipette to resuspend nuclei in the Tagmentation Master Mix with amulti-channel pipettor.

2.

Incubate the tagmentation reaction at 37°C for 30 minutes in a thermomixer (or similar
device) set at 800 rpm.

3.

Immediately following the tagmentation reaction, transfer each sample to a clean 1.5
mL microcentrifuge tube.

4.

Add 250 µL DNA Purification Binding Buffer and 5 µL 3 M sodium acetate toeach sample.

5.

If the color of the sample is anything other than bright yellow, add additional 3 M
sodium acetate in 5 µL increments until the proper color is achieved. Please see

the figure below.
Figure 1: Solution color as a function of pH.

The DNA Purification Binding Buffer has a pH indicator dye, so that the pH of thesolution can easily be determined. Only apply the sample to the column when thesolution is bright yellow (tube on the left), indicating a pH under 7.5. DNA will not bindto the column if the pH is higher than 7.5.
6.

Mix each sample by pipetting.

7.

For each sample, place a labeled DNA purification column into a collection tube.

8.

Transfer each sample to its corresponding column, close the cap, and centrifuge at
17,000 x g (14,000 rpm) for 1 minute.

9.

Discard the flow-through and return the collection tube to the column.

Note:

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

10. Add 750 µL of Wash Buffer (see note above in step 9 the Wash Buffer should have a
final concentration of 80% ethanol) to the column and cap the column.
11. Centrifuge at 17,000 x g for 1 minute.
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612. Discard the flow-through and return the column to the collection tube.
13. With the column cap open, centrifuge at 17,000 x g for 2 minutes to remove residual
Wash Buffer from the column.
14. Transfer each column to a new microcentrifuge tube.
15. Add 35 µL of DNA Purification Elution Buffer to the center of the column matrix, cap
the column, and incubate for 1 minute at room temperature.
16. Centrifuge at 17,000 x g for 1 minute.
17. Discard column. DNA purification is complete.
18. Purified DNA can be stored at -20°C, or you may proceed directly with the PCR
Amplification of Tagmented DNA steps that follow.

PCR Amplification of Tagmented DNA
Note:
1.

If a kit such as the KAPA Real-Time Library Amplification Kit is used, an initial
72°C extension step is essential.

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 aunique 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 use one i7 Indexed Primer and use one i5 Indexed Primer:
i7 Indexed Primer

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

Reagents

VolumeTagmented DNA

33.5 µL

i7 Indexed Primer (25 µM)

2.5 µLi5 Indexed Primer (25 µM)

2.5 µLdNTPs (10 mM)

1 µL

5X Q5 Reaction Buffer

10 µL

Q5 Polymerase (2 U/µL)

0.5 µL

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2.

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

3.

Perform SPRI clean-up with 60 µL SPRI bead solution (1.2X the sample volume),
eluting in 20 µL DNA Purification Elution Buffer. Have 400 µL of freshly-prepared 80%
ethanol ready per sample:
A. Add 60 µL well-mixed, room temperature SPRI Beads to each sample.
B. Vortex briefly to mix and incubate for 5 minutes at room temperature to allow
beads to bind.
C. Apply magnet to collect beads.
D. Once the solution is clear, aspirate the supernatant.
E. With the magnet still applied to the sample, add 180 µL 80% ethanol to eachsample 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.
Once the beads transition from shiny to matte (2-5 minutes), proceed to thenext step.
J. With the tubes separated from the magnet, add 20 µL DNA Purification
Elution Buffer.
K. Cap tubes and vortex to mix.
L. Incubate samples for 5 minutes at room temperature.
M. Apply magnet to collect beads.
N. Once the solution is clear, transfer each supernatant containing the eluted DNA toa fresh tube.

4.

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

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

Sample SheetN701

TCGCCTTATAAGGCGA

N702

CTAGTACGCGTACTAG

N703

TTCTGCCTAGGCAGAA

N704

GCTCAGGA

TCCTGAGC

i5 Indexi5 Sequence

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

N501

TAGATCGCTAGATCGC

N502

CTCTCTATCTCTCTAT

N503

TATCCTCTTATCCTCT

N504

AGAGTAGAAGAGTAGA

i5 Indexi5 Sequence

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

N501

TAGATCGCGCGATCTA

N502

CTCTCTATATAGAGAG

N503

TATCCTCTAGAGGATA

N504

AGAGTAGATCTACTCT

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

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9References
1.

Buenrostro, J. D., et al. (2013) Nat. Methods 10: 1213-1218.

2.

Adley, A., et al. (2010) Genome Biol. 11.

3.

Corces, M. R., et al. (2017) Nat. Methods 14: 959-962.

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10Troubleshooting Guide
Problem/Question

Possible cause

Recommendation

High background insequencing data

Cell viability may be theissue. Apoptotic cells
release unprotected
DNA that is much moreaccessible to Tn5 thanDNA in compacted
chromatin, leading to highbackground signal.

Optional treatment with DNase I can improve results.
This treatment is only an option of cells that are viable
and able to exclude the enzyme3.

No library produced

Sample loss is possible
throughout the protocol,
especially when working
with small numbers ofcells as the pellet may
be difficult or impossible
to see.

Be sure to orient tubes so you know where the cellpellet will be, and pipette from the side of the tube
that is opposite from the pellet in all centrifugation
steps except for the removal of lysis buffer where a few
microliters of supernatant can be left behind.

Incompatible amplification
program used.

An initial 72°C extension step before denaturation
is essential for ATAC-Seq libraries because the 5’
ends of adapters’ non-transferred strands are notligated to insert DNA by the enzyme. The extension istherefore required in order to produce the anchor site
for the index primers. There is no real recovery from
this, unfortunately.

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11Frequently Asked Questions
Question

AnswerIs this kit the same as theoriginal Buenrostro et al.
Nature 2013 paper?

This kit is based off a combination of the original Buenrostro paper and theCorces et al. Nature 2017 paper, and we have optimized the buffers for amore robust and efficient assay.

What is the source of therecombinant Tn5 enzyme inthis kit?

Be sure to orient tubes so you know where the cell pellet will be, and pipettefrom the side of the tube that is opposite from the pellet in all centrifugation
steps except for the removal of lysis buffer where a few microliters ofsupernatant can be left behind.

What is the minimum
number of cells or tissue
per reaction?

50,000 cells or 20 mg tissue.

Can frozen tissues and cells
be used?

Yes, but the samples must be high quality to preserve viability. Cells shouldbe cryopreserved in a controlled-rate freeze with media formulated to protect
against ice crystal formation and subsequent cell damage, and tissue should
be flash frozen at -80°C.

Can nuclei be frozen forlater use in ATAC-Seq?

Yes, extracted nuclei can be cryopreserved and frozen at -80°C for later use.
Nuclei can also be pooled together to increase the sample size.

Can FACS-sorted cells
be used?

It is possible, but cells could be damaged during the FACS-sorting process.
For good ATAC-Seq libraries, cells must be viable.

Do you have a
recommendation for a 40
micron mesh strainer fortissue samples?

Yes, we recommend the Falcon 40 micron cell strainer (Falcon catalog
no. 352340).

Is DNase treatment
recommended forall samples?

DNase treatment can help in certain situations such as mitochondrial DNA
depletion, or if there are 15% dead cells in the sample, but it can end up
removing cells you want data from if the cells aren’t healthy and able toexclude the enzyme. It’s only recommended as an option when cells are
viable and won’t also be digested by the enzyme.

I don’t have a thermomixer.
Can I still use the kit?

Using a thermomixer is recommended. However, we have tested this andincubated the tagmentation reaction without the use of a mixer at 37°C in athermal cycler with a heated lid and the reaction was successful.

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

Everything except the Assembled Transposomes and Q5 DNA polymerase
can be thawed at room temperature. The Assembled Transposomes
and Q5 DNA Polymerase are formulated with glycerol and will not needthawing, 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 bedone simultaneously.

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Question

AnswerWhat is the expected final
Library fragments should range from around 250 bp to 1000 bp in length withlibrary size, and what should an oscillation period ~150 bp. To see example traces, please visit the kit web
the library traces look like on page at activemotif.com/atac-seq-kit
a fragment analyzer?
How many sequencing reads Typically 30 million paired-end reads is sufficient, with 20 million being a
are needed per sample?
minimum. The number of reads is dependent both on the sample genome
and the end goal of the ATAC-Seq assay. If the sample genome is verylarge, or if more advanced analysis is required, more sequencing reads may
be needed.
Can I multiplex more than
16 samples using the
ATAC-Seq Kit?

The ATAC-Seq Kit is supplied with 4x4 unique dual indexes for 16 uniquesamples. The indexed primers in the kit are identical to the Illumina Nextera
primers corresponding to N701-N704 and N501-N504. If you would like tomultiplex 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
the same concentration (25 µM) as those in the kit.

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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

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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