Enabling Epigenetics ResearchPre-indexed Assembled
Tn5 Transposomes
Catalog No. 53152(Version A3)

Copyright 2023 Active Motif, Inc.

Revision

Date

Description of Change

A2December, 2023

Added "High Fidelity DNA polymerase" to the Additional Materials
Required section on page 3

A3

August, 2024

Corrected error in table on page 2

Information in this manual is subject to change without notice and does not constitute acommitment 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
Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Kit Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  2Protocol  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  3
Additional Materials Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  3
Pre-indexed Assembled Tn5 Transposomes ATAC-Seq Assay Protocol  . . . . . . . .  4
Technical Services . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  6

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BackgroundPre-indexed Assembled Tn5 Transposomes are provided in a breakaway 96-well plate format
to enable higher throughput ATAC-Seq assays. By having a unique i7 & i5 Indexed Primer
combination in each well, it is easy to perform multiple ATAC-Seq reactions without having
to pipette primers separately and keep track of the combinations. The ATAC-Seq Buffer Set(Cat. No. 53153) is a convenient set of buffers to use for 96 ATAC-Seq reactions with the Preindexed Assembled Tn5 Transposomes.
How does the ATAC-Seq Assay work with the Pre-indexed Assembled Tn5 Transposomes?
Simply add 50,000 to 100,000 prepared cells per well, and incubate with Tagmentation
Master Mix for 30 minutes. Then, stop the reaction and collect tagmented DNA using SPRI
beads and purify the DNA and PCR amplify. The libraries are then ready for sequencing onan Illumina® Sequencing System. P5 and P7 Primers are included with the Pre-indexed
Assembled Tn5 Transposomes for post tagmentation PCR amplification.
ProductFormat

Catalog No.

Pre-indexed Assembled Tn5 Transposomes

1 x 96 rxns

53152

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Kit Components
Reagents

QuantityStorage

1 x 96 rxns

-20°C

P5 Primer (25 µM)

250 µL-20°C

P7 Primer (25 µM)

250 µL-20°C

Pre-indexed Assembled Tn5 Transposomes

Note:

The Pre-indexed Assembled Tn5 Transposomes must be stored at -20°C.
Improper storage results in poor ATAC-Seq assay results. The Pre-indexed
Assembled Tn5 Transposomes are formulated in glycerol and are stablethrough quick freeze-thaw cycles when breaking away the wells for use in anassay. Be sure to store remaining wells at -20°C.

This product is guaranteed for six months from date of receipt when components arestored properly.
WellLocation

123456789101112Index

N505

N506

N507

N508

N510

N511

N513

N515

N516

N517

N518

N520

A

N701

N701,
N505

N701,
N506

N701,
N507

N701,
N508

N701,
N510

N701,
N511

N701,
N513

N701,
N515

N701,
N516

N701,
N517

N701,
N518

N701,
N520

B

N702

N702,
N505

N702,
N506

N702,N507

N702,
N508

N702,
N510

N702,
N511

N702,
N513

N702,
N515

N702,
N516

N702,
N517

N702,
N518

N702,
N520

C

N703

N703,
N505

N703,
N506

N703,
N507

N703,
N508

N703,
N510

N703,
N511

N703,
N513

N703,
N515

N709*,
N516

N703,
N517

N703,
N518

N703,
N520

D

N704

N704,
N505

N704,
N506

N709*,
N507

N704,
N508

N704,
N510

N704,
N511

N704,
N513

N704,
N515

N704,
N516

N704,
N517

N704,
N518

N704,
N520

E

N705

N705,N505

N705,
N506

N705,
N507

N705,
N508

N705,
N510

N705,
N511

N705,
N513

N705,
N515

N705,
N516

N705,
N517

N705,
N518

N705,
N520

F

N706

N706,
N505

N706,
N506

N706,
N507

N706,
N508

N706,
N510

N706,
N511

N706,
N513

N706,
N515

N706,
N516

N706,
N517

N706,
N518

N706,
N520

G

N707

N707,
N505

N707,
N506

N707,
N507

N707,
N508

N707,
N510

N709*,
N511

N707,
N513

N707,
N515

N707,
N516

N707,
N517

N709*,
N518N707,
N520

H

N708

N708,
N505

N708,
N506

N708,
N507

N708,
N508

N708,
N510

N708,
N511

N708,
N513

N708,
N515

N708,
N516

N708,
N517

N708,
N518

N708,
N520

Figure: Pre-indexed Assembled Tn5 Transposomes Indexed Primer Plate Map
Each well contains 4 µL of 8 µM Pre-indexed Assembled Tn5 Transposomes. This concentration isa combination of the indexed primer pairs and Tn5 (8 µM Tn5 + 4 µM oligo A + 4 µM oligo B). The
Pre-indexed Assembled Tn5 Transposomes have been validated in ATAC-Seq using 50,000 cells. Alli5 and i7 index combinations were tested to ensure efficiency in a biologically relevant tagmentation
reaction. i5 and i7 index combinations that were identified to produce less sequencing reads werereplaced with index N709 and normal sequencing reads resulted after re-testing to confirm theyyield optimal results. Wells where N709 replaced the index in use for other wells in that row arehighlighted with red N709* in the plate map above to easily see the changes in the logical layout ofthe plate.

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Protocol
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.

Additional Materials Required•

Micropipette and appropriate tips•

ATAC-seq Buffer Set (Cat. No. 53153) or 1% Digitonin, 2X Tagmentation Buffer,and ATAC Lysis Buffer•

10X PBS•

10% Tween20•

50,000-100,000 nuclei•

Nuclease-free water•

Thermal shaker for PCR tubes•

DNA purification column clean up or 0.5M EDTA, 10% SDS and SPRI beads•

Thermal cycler•

High Fidelity DNA polymerase

activemotif.com|3Pre-indexed Assembled Tn5 Transposomes ATAC-Seq Assay Protocol
1.

Count cells and aliquot 50,000 to 100,000 cells into a fresh 1.5 ml centrifuge tube, or96-well plate for each sample.

2.

Centrifuge the cells at 500 x g for 5 minute at 4°C. If there is no visible pellet, spin anadditional 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.

Immediately spin down at 500 x g for 10 minutes at 4°C. During this time, prepare theTagmentation Master Mix.

6.

Prepare the following Tagmentation Master Mix and keep on iceIngredient

Per Sample Volume

2X Tagmentation Buffer

25 µL

10X PBS

2 µL

1% Digitonin

0.5 µL

10% Tween20

0.5 µL

Nuclease-free Water

18 µL

Total

46 µL

7.

After the spin, remove the supernatant very carefully, ensuring the cell pellet is notdisturbed (this is a critical step; take extra care to remove the ATAC Lysis Buffer but
not lose nuclei).

8.

Immediately resuspend each nuclei sample in 46 µL of the Tagmentation Master Mix
prepared in the previous step. Keep on ice.

9.

Retrieve 96-well Pre-indexed Assembled Tn5 Transposomes plate from -20°C andtear away the number of strips or wells desired. Promptly freeze any unused wells
at -20°C.

10. Quickly, but gently, spin down the strips or plate in a centrifuge to collect all liquid atthe bottom of the well.
11. Working with the plate or strip wells containing the Pre-indexed Assembled Tn5
Transposomes in a cold block or on ice, remove the strip caps and add resuspended
nuclei from step 2 directly to the well. Be sure to keep track of the orientation of thecaps so the cap returns to the correct tube.
12. Re-cap the wells, gently vortex, and quick spin.
13. Incubate Pre-indexed Assembled Tn5 Transposomes plate or strip wells containing
nuclei and Tn5 at 37°C with shaking at 800 rpm for 30 minutes.

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414. After the incubation, stop the tagmentation reaction using method A or B:
A. EDTA and SPRI Beads
i. Add 1.8 µL of 0.5M EDTA and 0.5 µL of 10% SDS directly to thetagmentation wellii. Re-cap, vortex to mix, quick spin, and incubate the sample at 55°C for3 minutesiii. Collect tagmented DNA using desired SPRI bead ratio (1.2X ratiois recommended)
B. Purify the sample on a DNA purification column following the column
specific protocol.
At this point, purified, tagmented DNA fragments can be prepared by PCR amplified for next
generation sequencing on an Illumina® Sequencing System. The PCR amplification primers
included are supplied as 25 µM in TE buffer. Using the primers at a final concentration of 1.25
µM in the PCR reaction is recommended (2.5 µL of each primer per 50 µL reaction).
The following PCR protocol is recommended:
Note:

The number of cycles is based on starting with 50,000 – 100,000 nuclei. This
step will need to be optimized if more or less starting material is used.

72°C for 5 minutes
98°C for 30 seconds
8-10 cycles of:
98°C for 10 seconds
63°C for 30 seconds
72°C for 1 minuteHold at 10°C
For index primer and sample sheet information, visit the Pre-indexed Assembled Tn5
Transposomes web page at activemotif.com

activemotif.com|5Technical Services

ChinaJapanEurope

North 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

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760 431 1351

E-mail:tech_service@activemotif.com

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