TECHNOTE
Enabling Epigenetics Research

PIXUL® Sonicator and Agilent Electrophoresis
Systems for Chromosome Configuration Analysis
Advantageous combination of PIXUL® Multi-Sample Sonicator from Active
Motif with Automated Electrophoresis systems from Agilent Technologies tostudy chromosome configuration using Hi-C

For Research Use Only. Not for use in diagnostic procedures.
Agilent Technologies PR7001-2593

TECHNOTE:PIXUL® Sonicator and Agilent Electrophoresis
Systems for Chromosome Configuration Analysis

Introduction
Knowledge of epigenetics helps to advancetherapeutic innovation and personalized medicine as a regulator of health, development,and disease. An important contributor to epigenetic regulations is the effect of long-range
chromosomal interactions between distantgenomic loci that regulate 3-dimensional (3D)
configuration of chromatin, gene expression,and the participation of associated transcription factors. Long-range chromosomalinteractions are dynamic, and changes canoccur during developmental stages, disease
conditions, drug treatment, and other physiological events, thereby impacting epigenetic
regulations. These long-range interactions
need to be studied because of their tremendous biological significance. The Hi-C assay
is an efficient method for unbiased detection of chromosomal interactions and their
3D conformation. The data from Hi-C analysis
provides significant insights into understanding gene regulatory networks and predicting
transcription factor activity.
Like most epigenetic methods, the Hi-C
workflow requires significant time, labor, andexpenditure. As this assay has become morewell-known, new tools have been developed
to address workflow difficulties. Two major
challenges to a successful Hi-C workflow areto obtain optimum DNA fragmentation withina precise range, and to obtain good quality of
the final Hi-C library. The first challenge arises
from inconsistencies in existing methods ofDNA fragmentation, often due to inefficientmethods, incompatibility with high-throughput platforms and high costs. Inconsistency
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in sizes of DNA fragments is a major contributor to the second challenge, obtaining ahigh-quality final Hi-C library. A poor-quality
library leads to experimental failure or sub-optimal results and lost resources.
The first challenge of consistent DNA fragmentation is successfully addressed by thePIXUL® Multi-Sample Sonicator from Active
Motif, which offers consistent, user-friendly, fast, and high-throughput sonication of96-samples using an inexpensive microplate.
Successful fragmentation of the samples
should be confirmed by the Agilent automatedelectrophoresis systems at this step to ensure
samples are within an optimal size range tofurther proceed with the Hi-C workflow.
The second challenge of obtaining high quality
NGS (next-generation sequencing) libraries isaided by again using the Agilent automatedelectrophoresis systems for quality control,
allowing for the identification of libraries
that are high quality for sequencing or that
may need further optimization. The Agilent
systems perform accurate sizing and quantification of up to 96 samples at a time using
only 1-2 µL per sample in a single run, whilestill maintaining a streamlined user-friendly
workflow. By providing reliable DNA fragmentation and quality control, the combinationof these technologies in the Hi-C workflow
alleviates major challenges and contributes
to overall experimental success.

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TECHNOTE:PIXUL® Sonicator and Agilent Electrophoresis
Systems for Chromosome Configuration Analysis

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

Experimental Setup for
PIXUL and Agilent AutomatedElectrophoresis Systems
Active Motif uses the Hi-C portfolio from Arima
Genomics. The Hi-C workflow performed at
Active Motif is schematically represented inFigure 2. First, samples are collected in theform of cells or tissue. Next, chromatin in thesamples is crosslinked and fragmented, andthe ends are repaired and biotinylated. The
DNA is then ligated at proximal regions ofgenomic interactions as shown in step 5 ofFigure 2. Ligated DNA is subjected to sonication where 100 µL of sample is loaded into
each well of a 96-well round bottom plate(Corning 3799) which is sealed and loaded
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onto PIXUL® where the following sonication
program is set – Pulse (N): 50, Pulse Rate
Frequency: 1 kHz, Burst Rate: 20 Hz, Process
Time 40 minutes. The program can be optimized as needed, with recommendationsavailable upon request.
After sonication in step 6 of Figure 2, the96-well plate is removed from PIXUL®, andcentrifuged for ~10 seconds at 500 rpm forcollecting all content into the wells. Next,
the size of the fragmented DNA is confirmed
by a quality control (step 7, Figure 2) using
one of the Agilent automated electrophoresis systems, either the TapeStation or theFragment Analyzer, and the associated kits.
Both systems have multiple reagents and kits
for nucleic acid analysis covering expansiveJapanjapantech@activemotif.com

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TECHNOTE:PIXUL® Sonicator and Agilent Electrophoresis
Systems for Chromosome Configuration Analysis

Crosslink
chromatin

Cells or tissue

Ligate fragmented
chromatin

9Hi-C librarygeneration

Fragment
chromatinSonication
by PIXUL®

10Repair andbiotinylate ends

Quality control analysis
by Agilent System

Quality control analysisof Hi-C library

118Pull-down
biotinylated DNA

Paired-end sequencing
and bioinformtics analysis

Figure 2. Active Motif Hi-C service workflow. Sonication by the PIXUL® is performed instep 6. The Agilent automated electrophoresis systems, either the TapeStation or theFragment Analyzer, are used for quality control of the fragmented sample and of the final
Hi-C library, as shown in steps 7 and 10, respectively.

sizing and concentration ranges. The sample
quality is considered acceptable when theoutput appears as the smear shown in Figure
3 with the largest portion of the smear around
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400 base pairs. The experiment will thenproceed to immunoprecipitation and Hi-C
library preparation (steps 8 and 9, Figure 2).

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TECHNOTE:PIXUL® Sonicator and Agilent Electrophoresis
Systems for Chromosome Configuration Analysis

The final Hi-C libraries undergo quality control
using the Agilent TapeStation or Fragment
Analyzer (step 10, Figure 2) for fast andaccurate analysis of sample size distribution,quantification, and purity. This quality control
check is crucial to determining whether thelibraries can proceed to sequencing and bioinformatics analysis (step 11, Figure 2).

Results
Prior to immunoprecipitation, DNA fragmentssonicated by PIXUL® are analyzed usingAgilent automated electrophoresis systems
for accurate quantification and sizing assessment as a quality control step. Figure 3 represents an example of the analysis of PIXUL®sonicated samples using the TapeStation.
The 3 lanes of the electronic gel in Figure

3 show the sizes of DNA fragments from 3
samples sonicated by PIXUL®. TapeStation
data can be presented as either a digital gel
image, or as individual electropherograms.
Additionally, a data table shows informationsuch as the concentration and average size ofeach sample. The example shown in Figure 3
demonstrates the fragmented pattern expected for PIXUL sonicated samples in the Hi-Cworkflow. Accurate analysis of the samples
by TapeStation confirms that the samples
have sufficiently met Active Motif’s DNA
fragment size and consistency standards asshown in Figure 3 where the largest portionof the smear appears around 400 basepairs. Samples which pass the crucial quality
control steps using the Agilent systems
proceed to immunoprecipitation, followed bylibrary preparation.

Figure 3. Agilent TapeStation analysis after PIXUL® sonication of 3 samples, prior topull-down of biotinylated DNA. The electropherogram and region table correspond to thesample in lane 1 of digital gel. Blue lines on electropherogram indicate region analysis
settings. Region table shows average size, concentration, and percent total of portion ofsample within the user-specified region.
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TECHNOTE:PIXUL® Sonicator and Agilent Electrophoresis
Systems for Chromosome Configuration Analysis

Figure 4. Agilent TapeStation analysis of Hi-C libraries. The electropherogram and regiontable correspond to the sample in lane 1 of the digital gel image.

The Agilent automated electrophoresis
systems can then be utilized a second timein the Hi-C workflow for quality control of thefinal NGS library prior to sequencing. Figure
4 shows TapeStation analysis of final Hi-C
libraries. The 3 lanes of the electronic gel in
Figure 4 are the final Hi-C libraries generated from the same 3 samples represented in
Figure 3. Hi-C libraries with size distributions
and patterns similar to Figure 4, qualify as
passing. Libraries which do not pass quality
control should not proceed to sequencing
and risk a failed experiment or incorrect interpretation of results, along with loss of precious resources invested in the workflow. TheAgilent automated electrophoresis systems
allow the user, in this case Active Motif, tomake crucial decisions regarding the courseof the experiment.

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Figures 3 and 4 provide examples of the dataassessment provided by TapeStation systems
in the Hi-C workflow. Alternately, the Agilent
Fragment Analyzer can also be used toexamine fragmented DNA and Hi-C libraries
using the ProSize data analysis software (datanot shown).

Conclusion
Hi-C helps to detect epigenetic effectors likechromosomal conformation and 3-dimensional interactions between genomic loci which
can be analyzed in healthy versus diseasestates, various stages of development, drug
treatment, and other physiological events.
As these scenarios are often studied within
disease research and therapeutic development, Hi-C is a valuable assay within therapeutic and pharmaceutical industries.

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TECHNOTE:PIXUL® Sonicator and Agilent Electrophoresis
Systems for Chromosome Configuration Analysis

This application note demonstrates theimportance and benefits of integrating thePIXUL® and Agilent automated electrophoresis systems into the Hi-C workflow. Sonication
by PIXUL® provides consistent DNA fragmentation, which is necessary for downstream
processing. The automated electrophoresis
systems are integral to crucial quality control
steps throughout, including confirming thesize of the fragmented DNA sample andthe quality of the Hi-C NGS library before
sequencing. Both PIXUL® and Agilent systems
are compatible with high-throughput workflows as they improve the simplicity andspeed of the protocol with increasing accuracy and precision.
Additionally, the PIXUL and Agilent systems
make optimization of the Hi-C workflow
easier. This is because PIXUL® allows up to 12
different sonication programs to be assigned
to the 12 columns of a 96-well microplate forsimultaneous processing, making it possible
to test for multiple sonication conditions in asingle run. Afterwards, the high-throughput
samples can be prepared in a simple mannerand rapidly analyzed with the TapeStation.
The Fragment Analyzer can also be used for
precise analysis of high-throughput samples
because it allows up to three 96-well plates
to be loaded and analyzed without userintervention. Both Agilent systems require
only 1-2 µL of sample, minimizing sample
loss. Together, a combination of PIXUL® andAgilent automated electrophoresis systems
significantly shorten sample optimization timeand assures successful Hi-C sequencing.

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Further details and advantages ofTapeStation and Fragment Analyzer can befound here at agilent.com
TapeStation
Fragment Analyzer
For more information on thePIXUL® Multi-sample Sonicatorvisit activemotif.com/pixul
PIXUL®

byRwik Sen, Ph.D.,
Samuel Mayorga, B.S.,
Ryan Kostecki, B.S.,
Samantha Cassi, B.S.,
Zarina Gallardo, B.S.

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