TECHNOTE
Enabling Epigenetics Research

Use of PIXUL® Multi-Sample Sonicator with
Agilent Automated Electrophoresis for ChIP-Seq
Targeted high-throughput epigenetics analysis using ChIP-Seq is facilitatedby combining PIXUL® Multi-Sample Sonicator from Active Motif with theAgilent Automated Electrophoresis Portfolio

TECHNOTE:

Use of PIXUL® Multi-Sample Sonicator with
Agilent Automated Electrophoresis for ChIP-Seq

Introduction
Epigenetics answers significant questions
regarding personalized medicine and avariety of diseases. Several challenges toepigenetics research include low sample
quantity and sample preparation. Traditional
epigenetic methodologies generally
require large amounts of sample, which
can be difficult to obtain from patients, andworkflows involve several sample transfer
steps resulting in sample loss. Additionally,consistent, quick, efficient, robust, and costeffective preparation of high-throughput
samples and their downstream analysis
continue to pose significant challenges. To
address some of the challenges associatedwith sample preparation, the PIXUL® MultiSample Sonicator from Active Motif canbe combined with the Agilent automatedelectrophoresis systems, like the Fragment
Analyzer and the TapeStation (Figure 1).
The PIXUL provides rapid and consistentsample fragmentation through sonication,
while the Agilent systems provide accurate
sizing and quality metrics of the fragments
as crucial aspects of next-generation
sequencing workflows.
In a single run, PIXUL offers the flexibility
to process 1 to 96 samples in a 96-wellplate where 12 columns of that plate can be
individually programmed. Its setup and run
times are quick, and output is consistent.
Both the Fragment Analyzer and TapeStation
systems can be used for reliable evaluationof the sonicated material and the final NGSlibrary, providing accurate sizing which is a
crucial quality control step. If the analysis
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indicates that the size distribution of thesonicated fragments needs to be altered,then the flexibility of the PIXUL makes theoptimization process quick and convenient
to identify best sonication parameters for a
specific assay. The versatility of the PIXULand the automated electrophoresis systems
makes them adaptable to a variety ofworkflows across multiple omics realms.
In epigenetics, chromatin
immunoprecipitation and sequencing (ChIPSeq) is a popular method which provides
genome-wide locations of interaction
between a specific protein or histone
modification and chromatin/DNA. ChIPSeq is widely used in various fields of
biological and biomedical research. Being animportant, laborious, and expensive method,
ChIP-Seq needs crucial quality control in
its workflow. Hence, this Technical Notefocuses on the combination of PIXUL with
Agilent’s Fragment Analyzer and TapeStation
for crucial quality control in ChIP-Seq
library preparation.

ChIP-Seq Workflow
ChIP-Seq maps genome-wide localizationof a protein or histone modification ofinterest. Hence, crosslinking is performed
for immobilization of chromatin-interacting
proteins on DNA. Since the actual length ofchromatin/DNA strands are not conduciveto the ChIP-Seq workflow, they need tobe fragmented down to compatible sizes.
Hence, crosslinked samples undergo
sonication to generate fragments around
300 base pairs, ideal for ChIP-Seq.
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TECHNOTE:

Use of PIXUL® Multi-Sample Sonicator withAgilent Automated Electrophoresis for ChIP-Seq

Figure 1: From left – PIXUL®, Agilent 4200 TapeStation system, and Agilent 5300
Fragment Analyzer system

Next, the fragments undergo
immunoprecipitation (IP) using an antibody
against the protein of interest which has
been initially crosslinked to chromatin/
DNA. An aliquot of fragmented chromatin
is not subject to IP and serves as an input
control. The DNA which is obtained by IP
and the input DNA are purified, amplified,and sequenced. The sequencing resultsfrom the immunoprecipitated DNA and input
control are compared to determine thesuccess of ChIP-Seq and to interpret theresults. An outline of the ChIP-Seq workflow
is presented in Figure 2.
Two crucial steps in ChIP-Seq are 1) DNA
sonication for generating consistent
fragment sizes, and 2) antibody specificity.
While several solutions regarding antibody
improvement are commercially available,
the critical criterion of generating consistent
fragment sizes is not well-explored.
Inconsistent and improper sonication causes
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ChIP-Seq to either fail or to provide incorrect
and misleading information. Issues with thiscrucial step in the ChIP-Seq workflow areeasily resolved with the PIXUL sonicatorfrom Active Motif, which consistently
fragments chromatin/DNA down to sizes thatare ideal for ChIP-Seq. Following sonication,
the Agilent automated electrophoresis
systems enable precise analysis of fragment
sizes, a pivotal quality control step that helps
to decide the course of the workflow. If thefragments are of the appropriate size, thesample proceeds through library preparation
as shown in Figure 2. The Agilent automatedelectrophoresis systems can be utilized asecond time in the ChIP-Seq workflow for
quality control analysis of the final libraryprior to sequencing. The combination of thePIXUL and the automated electrophoresis
instruments helps ensure the generation ofconsistent fragment sizes and successful
ChIP-Seq results.

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TECHNOTE:

Use of PIXUL® Multi-Sample Sonicator with
Agilent Automated Electrophoresis for ChIP-Seq

Chromatin Preparation

PIXUL®
• Harvesting• Crosslinking
Sonication

Chromatin
Immunoprecipitation

Input

Fragment
Analyzer

No antibodytreatment

DNA Purification

TapeStation

orQC: Analysis of Sonicated Chromatin

ΔRxn

ChIP DNA Validation

Cycle

Sequencing Library Construction• Perform End Repair• Add ‘A’ Overhangs to 3´ Ends
• Ligate Adapters• Size Select the Library• Enrich by PCR

Fragment
Analyzer

TapeStation

orValidate the Library

Sequencing

QC: Analysis of Libraries

Figure 2: ChIP-Seq workflow, indicating the steps where the PIXUL®, the Agilent 5300
Fragment Analyzer system, and the Agilent 4200 TapeStation system are used. PIXULis used for sonication of chromatin into fragments whose sizes are compatible withChIP-Seq. The Agilent equipment are used to quantify the sizes of the sonicatedfragments and later for ChIP-Seq library quality control analysis.
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TECHNOTE:

Use of PIXUL® Multi-Sample Sonicator with
Agilent Automated Electrophoresis for ChIP-Seq

Materials and Methods
To sonicate samples for ChIP-Seq using
PIXUL, crosslinked chromatin is resuspended
in lysis buffer and added in each well of a
96-well round bottom plate, which is thensealed carefully (Active Motif Catalog No.
53139 or Corning Catalog No. 3799). The
sealed plate is loaded into PIXUL as per
instructions in the PIXUL manual. The PIXUL
touchscreen is operated for the next setof actions.
On the PIXUL touchscreen, Circulation isturned on to maintain low temperatureduring fragmentation, and to remove any
bubbles which may appear underneath theexternal surface of the 96-well plate uponloading it into PIXUL. The touchscreen isthen used to set the sonication programfor 1 to 12 columns of the 96-well plate andto Start sonication. For DNA fragmentation
the following simple parameters are used –Pulse (N): 50, Pulse Rate Frequency: 1 kHz,Burst Rate: 20 Hz, Process Time: optimized
as needed. Information on PIXUL protocols
for various applications can be foundhere. After sonication, the 96-well plate isremoved from PIXUL and centrifuged for~10 seconds to collect all fluids down to thebottom of wells.
The size of the sonicated DNA is assessedwith the Fragment Analyzer using thequalitative dsDNA 930 Reagent kit (Agilent
p/n DNF-930-K0500) according to themanufacturer’s instructions. This is one of
the most crucial quality control steps in theChIP-Seq workflow. The fragmented DNA isNorth America
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then subjected to the subsequent steps of
the ChIP-Seq workflow as shown in Figure
2. When the libraries are prepared, theFragment Analyzer is used again for quality
control analysis. Alternately, quality control
can also be performed using the Agilent
TapeStation (Figure 2).

Results
The size of fragmented DNA/chromatin
undergoing immunoprecipitation is crucial
to successful ChIP-Seq workflows. To
test whether PIXUL sonication fragments
the DNA into sizes ideal for ChIP-Seq, anaccurate method of analysis is implemented
using either the Fragment Analyzer (Figure3) or TapeStation (Figure 4). This analysis
helps researchers make the criticaldecisions about whether the fragments
qualify for downstream processing, such as
immunoprecipitation, library preparation,and sequencing.
Shown in this application note are examplesof the quality control steps in the ChIPSeq workflow where Agilent systems areimplemented. As shown in Figure 2, thefirst QC step is analysis of the sonicatedchromatin. Example of the results achievedat this QC step can be seen in Figures 3and 4. Analysis with the Agilent automatedelectrophoresis systems presents the results
as an electropherogram or a digital gel.
In this example, unsonicated and PIXULsonicated chromatin which is purified fromhuman brain samples are compared on theFragment Analyzer (Figure 3). Compared tothe sonicated DNA, the unsonicated sample
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TECHNOTE:

Use of PIXUL® Multi-Sample Sonicator withAgilent Automated Electrophoresis for ChIP-Seq

is characterized by the absence of lower
molecular weight DNA in the gel lane. Incontrast, the PIXUL sonicated sample yields
fragments around 300 bp, the ideal size
range for the ChIP-Seq workflow. Figure
4 shows a similar analysis of mouse brain
using the TapeStation, which also provides
accurate size analysis of sonicated DNA(Y) and very definitive contrast to theunsonicated control (N).

Once the size of the sonicated sample has
been confirmed, the ChIP-Seq workflow
continues through library preparation as
depicted in Figure 2. The sonicated samples
shown in Figures 3 and 4, were thus used
for immunoprecipitation of the sonicatedchromatin from human and mouse brain
using an antibody against histone H3lysine 9 acetylation (H3K9ac). ChIP-Seq
libraries were prepared and analyzed on theAgilent automated electrophoresis systems.
Representative examples of the input control
and the ChIP-Seq libraries generated fromthe immunoprecipitated DNA are shown on
the TapeStation (Figure 5).

Figure 3: PIXUL-sonicated and unsonicatedchromatin purified from an input prep using
human brain sample, analyzed on the Agilent
5300 Fragment Analyzer system.

Figure 4: PIXUL-sonicated (Y) and unsonicated(N) chromatin purified from an input prepusing mouse brain sample, analyzed on theAgilent 4200 TapeStation system.

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TECHNOTE:

Use of PIXUL® Multi-Sample Sonicator withAgilent Automated Electrophoresis for ChIP-Seq

Figure 5: ChIP-Seq libraries of immunoprecipitated (IP) DNA and corresponding input
DNA from human and mouse brain samples, analyzed on the Agilent 4200 TapeStation
system. IP using antibody against histone H3 lysine 9 acetylation (H3K9ac). Sizes (bluearrows) are higher than Figures 3 and 4 because these fragments are ligated withsequencing adapters during library preparation.

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TECHNOTE:

Use of PIXUL® Multi-Sample Sonicator with
Agilent Automated Electrophoresis for ChIP-SeqConclusion
The combination of PIXUL sonication andquality control analysis with the Agilent
automated electrophoresis portfolio, i.e.,
the Fragment Analyzer and TapeStation,
throughout the ChIP-Seq workflow
significantly contributes to its success. PIXULprovides consistent sizes of chromatin
fragments which is a necessity for ChIP-Seq.
The automated electrophoresis systems
enable quality control at two key steps ofsize analysis in the workflow: the sheared
DNA and the final library. Together, theuse of these systems leads to high quality
library preparation with very consistentsize distribution, which are ideal for ChIPSeq. Further details on the advantages tousing the Agilent Fragment Analyzer can befound here. Likewise, the TapeStation has
several advantages which are found here.
Overall, the PIXUL and Agilent automatedelectrophoresis systems provide importantsolutions towards successful execution ofcomplex, laborious, and expensive methods
like ChIP-Seq.
Agilent PR7001-1524
byRwik Sen, Ph.D. and Dana Meents, M.S.

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