PIXUL™ Chromatin Shearing Kit Manual
Catalog No. 53132(version A1)

Active Motif North America
1914 Palomar Oaks Way, Suite 150
Carlsbad, California 92008, USAToll free:877 222 9543
Telephone:
760 431 1263
Fax:
760 431 1351
Active Motif Europe
Waterloo Atrium
Drève Richelle 167 – boîte 4
BE-1410 Waterloo, Belgium
UK Free Phone:		
0800 169 31 47
France Free Phone:
0800 90 99 79
Germany Free Phone: 0800 181 99 10
Telephone:		+32 (0)2 653 0001
Fax:		
+32 (0)2 653 0050
Active Motif Japan
Azuma Bldg, 7th Floor
2-21 Ageba-Cho, Shinjuku-Ku
Tokyo, 162-0824, Japan
Telephone:+81 3 5225 3638
Fax:
+81 3 5261 8733
Active Motif China
787 Kangqiao Road
Building 10, Suite 202, Pudong District
Shanghai, 201315, China
Telephone:(86)-21-20926090
Hotline:400-018-8123
Copyright 2020 Active Motif, Inc.
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TABLE OF CONTENTS

Page

Overview . .  .  .  .  .  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Kit Components and Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Reagents for Chromatin Shearing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Additional Materials Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Protocol . .  .  .  .  .  .  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Technical Services  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

Information in this manual is subject to change without notice and does not constitute a commitment on thepart of Active Motif, Inc. It is supplied on an “as is” basis without any warranty of any kind, either explicit orimplied. 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 andprotected by the copyright laws of the United States and international treaties.
PIXUL™ is sold under an exclusive license to patents owned by Matchstick Technologies Inc. and University ofWashington, specifically Matula, T.J.; Bomsztyk, K.; Darlington, G.P., Maxwell, A.D.; MacConaghy, B.E., Reed, J.
“Ultrasound system for shearing cellular material”. US Patent US10809166. European Patent EP3169451.
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 their respective companies.
For Research Use Only. Not for use in diagnostic procedures.

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Overview
Highly consistent shearing of chromatin is critical for successful downstream chromatin
immunoprecipitation assays. Manual shearing with a probe sonicator or shearing with aninconsistent high-throughput sonicator can lead to sample-to-sample variability.
The PIXUL Chromatin Shearing Kit delivers highly consistent average fragment lengths across entire
plates time after time. This kit is compatible with a wide range of fixed cells (10,000 - 5,000,000)
with very low standard deviations in sonication efficiency.

PIXUL Advantages• Process 1 - 96 samples in parallel• Extremely consistent sonication• Up to 12 different sonication conditions per run
product
PIXUL™ Chromatin Shearing Kit

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1formatcatalog no.

1 X 96 reactions

53132

Kit Components and Storage
The kit contains sufficient reagents to sonicate 96 wells in one run. The reagents in this kit havemultiple storage temperatures. 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

QuantityStorage

Fixation Buffer

75 µL

4°C

Stop Solution

560 µLRT

10X PBS

6 mL

4°C

2 X 100 µL-20°C

Cell Shearing Buffer

11 mL

RT

PIXUL™ 96-well plate with sealer

1 unit

RTProtease Inhibitor Cocktail

Additional Materials Required• 100% Ethanol• Nuclease-free water• 16% methanol-free formaldehyde• 1X PBS• PIXUL Multi-Sample Sonicator (Cat. No. 53130)• Microcentrifuge tubes• PCR tube strips (300 µL well volume capacity)• Centrifuge to spin 96-well plate• Thermal cycler or other 37°C incubator• 96-well plate magnet or bar magnet• Single channel and multi-channel pipettors and tips
• Hemocytometer and light microscope

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PIXUL Chromatin Shearing Kit Protocol
IMPORTANT:PIXUL sonication requires the use of PIXUL 96-well round bottom plates. Theseplates are available as Active Motif Catalog No. 53139, and these are the sameplate as Corning Catalog No. 3799. Using any other plates will result in inefficientsonication, may damage the instrument, and will void the instrument warranty.

Buffer Preparation
Prepare the following buffers before starting the protocol.
1X PBS• 4.5 mL 10 X PBS• 40.5 mL nuclease-free water
Fixation Solution (Stock Solution)• 293 µL nuclease-free water• 90 µL Fixation Buffer• 867 µL 16% methanol-free formaldehyde
Fixation Solution in PBS (Working Solution)• 1 mL Fixation Solution (Stock Solution)• 10 mL 1X PBS
1X Stop Solution• 0.6 mL Stop Solution• 11.4 mL 1X PBS
Cell Shearing Buffer with Inhibitors• 10 mL Cell Shearing Buffer• 100 µL Protease Inhibitor Cocktail

Cell Fixation
1.

Culture cells directly in the PIXUL 96-well round-bottom plate or cell culture dish or flask ofyour choice.

2.

If you are using the PIXUL 96-well plates for cell culture, carefully aspirate media and add 100µL Fixation Solution (Working Solution).

3.

If you are not using the PIXUL 96-well plate for cell culture, collect cells, and resuspend theappropriate number of cells in 100 µL Fixation Solution (Working Solution) and transfer tothe PIXUL 96-well plate.

4.

Incubate for 10 minutes at room temperature. If you are working with suspension cells,collect cells by centrifugation.

5.

Aspirate Fixation Solution and add 100 µL 1X Stop Solution.

5.

Incubate for 5 minutes at room temperature. If you are working with suspension cells, collectcells by centrifugation.

6

Aspirate 1X Stop Solution and wash the cells twice with 100 µL 1X PBS. If you are workingwith suspension cells, collect cells by centrifugation before aspiration.
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PIXUL Sonication of Fixed Cells
1.

Aspirate PBS and add 100 µL Cell Shearing Buffer with Inhibitors to the fixed cells and seal
the plate with a PIXUL plate seal.
Note: All wells lacking sample in the columns being sonicated MUST be filled with liquid(water, coupling buffer, etc.) prior to starting the run.

2.

Turn on and run the instrument as specified in the PIXUL Multi-Sample Sonicator User
Manual.

3.

We recommend using the following parameter specifications as a starting point, and adjusting Process Time to optimize for your specific sample and application:
Labile marks, like phosphorylated epitopes, may be preserved better by discontinuous sonication (for example, rather than 30 minutes, 4 rounds of 4 - 6 minutes each, where the PIXUL
Coupling Fluid is allowed to circulate and cool between runs).
Sonication Parameter

Setting

Pulse [N]50

PRF [kHz]

1.00

Process Time [min]36:00

Burst Rate [Hz]

20.00

Note:

The instrument software will not allow settings that could damage thetransducers. The upper limits are a maximum Pulse of 50 or a maximum PRF
of 1.00. We recommend a minimum Burst Rate of 20.00. Varying the Process
Time is typically the only parameter that needs adjusting, with longer Timeneeded for difficult samples such as adipose tissue.

Analysis of Sonicated Chromatin
Remove 10 µL of the sonicated chromatin and use the PIXUL Chromatin Input Kit (Cat. No.
53134) to determine yield and sonication efficiency. We recommend shearing chromatin to200 - 500 bp in length for ChIP assays.

Using Chromatin Sheared Using PIXUL in ChIP Assays
Chromatin sheared with PIXUL is compatible with many ChIP protocols, but for best results
we recommend performing ChIP assays using the ChIP-IT High Sensitivity® Kit (Cat. No.
53040) or High-Throughput ChIP-IT® Kit (Cat. No. 53146).

www.activemotif.com4Technical Services
If you need assistance at any time, please call Active Motif Technical Service at one of thenumbers listed below.
Active Motif North America
1914 Palomar Oaks Way, Suite 150
Carlsbad, CA 92008, USA
E-mail: tech_service@activemotif.com
Active Motif Europe
Waterloo Atrium
Drève Richelle 167 – boîte 4
BE-1410 Waterloo, Belgium
E-mail: eurotech@activemotif.com
Direct: +32 (0)2 653 0001
Active Motif Japan
Azuma Bldg, 7th Floor
2-21 Ageba-Cho, Shinjuku-Ku
Tokyo, 162-0824, Japan
Active Motif China
787 Kangqiao Road
Building 10, Suite 202
Pudong DistrictShanghai, 201315, China

Toll Free: 877 222 9543
Direct: 760 431 1263
Fax: 760 431 1351

Germany Free Phone: 0800 181 99 10
France Free Phone: 0800 90 99 79
UK Free Phone: 0800 169 31 47
Fax: +32 (0)2 653 0050

Direct: +81 3 5225 3638
Fax: +81 3 5261 8733
E-mail: japantech@activemotif.com

Direct: (86)-21-20926090
Hotline: 400-018-8123
E-mail: techchina@activemotif.com

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