Enabling Epigenetics Research

TECHNOTE:TECHNOTE:

Sonicating Limited Tissue Samples
with PIXUL™ Multi-Sample Sonicatorfor ChIP-Seq Analysis

Sonicating Limited Tissue Samples with PIXUL™ Multi-Sample
Sonicator for ChIP-Seq Analysis

HeartLiver

KidneyLung

TECHNOTE:PIXUL & CHIP-SEQ WITH LIMITED AMOUNTS OF TISSUE SAMPLES
Introduction
Epigenetics has a huge potential in personalized
medicine as it becomes evident that it plays
a role in many diseases. However, preparing
samples in a clinical setting can be challenging.
With this TechNote we describe how to use thePIXUL™ Multi-Sample Sonicator to process tissue
samples for analysis using ChIP-Seq.

Challenges of Sample Processing from
Large Cohort Studies and Clinical Samples
Preparation of clinical samples for analysis can bechallenging for several reasons. Limited amountsof tissue are available with clinical samples, whichleads to a lack of sufficient material to answer
relevant questions in this setting. Due to thenature of clinical samples, chromatin preparationis complicated.
Furthermore, traditional methods of tissue
homogenization have limitations such as sample
loss and low amounts of cells being available,
which in turn yields a low amount of starting
material that can make some experiments
impossible. With such limitations from small
amounts of starting material and difficult-toprocess samples, reproducibility in data andconsistency in experiments is more important
than ever. Consistent shearing is necessary togenerate reproducible data.

Solution: PIXUL™ Multi-Sample Sonicator
In 2020, Active Motif launched the PIXUL™ MultiSample Sonicator, which delivers consistent, highthroughput shearing of cells, tissue or purified
biological samples with multiple parameters in one
run and on standard 96-well round bottom plates.
Each column in the 96-well plate is controlled byits own pair of transducers. As a result it is possible
to run up to 12 different optimization protocols at
one time, each column can run its own parameters.
With that, PIXUL enables the sonication of clinically
relevant numbers of samples simultaneously,
alleviating bottlenecks from traditional sonication
methodology.

Methodology and Data
So far 10 different human tissues have been testedon the PIXUL: kidney, liver, uterus, colon, adipose
tissue, ovary, PBMCs, heart, brain, and lung,providing a near to complete snapshot of relevant
tissue samples.

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We found that human tissues could be sonicatedon PIXUL in 72 minutes under a standard set ofparameters. We also tested 7 mouse tissues andfound that those can be sheared optimally in 36
minutes, indicating some difference in optimal
programs depending on species and tissue type.
With the ability of PIXUL to run 12 different programs
simultaneously, optimization of these programs persample type is easy.

Fixation of Primary Tissues
Primary tissues were harvested 24 hours post-mortem
and sent in preservation media to Active Motif.
Immediately upon arrival, tissues were washed 1x in
ice cold PBS, diced with a razor blade in PBS +4%
formaldehyde, moved to a conical tube and fixed for 15
minutes at room temperature with overhead rotation.
The reaction was quenched with 1/20th volume
2.5M glycine for 5 minutes at room temperature withoverhead rotation. Following fixation, samples werecentrifuged to remove the media before being snapfrozen in liquid nitrogen for long term storage at -80°C.

After homogenization, samples homogenized inCell Shearing Buffer without N-lauryl sarcosine werecentrifuged at full speed for 3 mins to pellet material
before resuspension in 1 mL Cell Shearing Buffer(+PIC/PMSF).

PIXUL™ Multi-Sample Sonication
After Bead Ruptor homogenization, 100 µL/wellof homogenized suspension were moved into
1 column (8 wells) of a 96-well round-bottom plateand sonicated on the PIXUL™ Multi-Sample Sonicator
at a Pulse [N] of 50, PRF [kHz] 1.00, Burst Rate [Hz]20.00, and Process Time [min] 72. After sonication,
the plate was spun at full speed for 1 min at 4°C
and the samples were pooled and aliquoted
into Eppendorf tubes for long term storage at-80°C. Sonicated material was compared with
unsonicated control after input DNA prep on
either Fragment Analyzer or TapeStation.

Analysis
1Bead Ruptor Homogenization
Small pieces of previously fixed tissue were moved
to a 2 mL Omni Bead Ruptor tube containing three
5 mm steel beads and 1 mL of either Cell Shearing
Buffer (+PIC/PMSF) or Cell Shearing Buffer without
N-laurylsarcosine (+PIC/PMSF). Kidney, PBMCs,liver, and brain were homogenized at 4 m/s for
20 sec. Lung, heart, ovary, uterus, adipose werehomogenized 2 for cycles of 4m/s for 20 sec. with~2 mins between cycles.

23Fig. 1: PIXUL Multi-Sample
Sonicator for Preparation ofDNA from Brain Tissue.
Fragmented chromatin from
10 different human tissue
samples was analyzed on a
Fragment Analyzer and comparedto unsonicated control.
Lane 1, DNA ladder.
Lane 2, unsonicated control sample.
Lane 3, sonicated brain tissue.

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TECHNOTE:PIXUL & CHIP-SEQ WITH LIMITED AMOUNTS OF TISSUE SAMPLESAs an example we show human brain below. Lane
1 shows the unsonicated control, lane 2 shows thesame sample sonicated for 72 minutes on PIXUL™
Multi-Sample Sonicator. The sonicated sample
shows an enrichment of fragments around 300
base pairs, which is the ideal size for ChIP-Seqanalysis.

After sonication, the plate was spun at full speed
for 1 minute at 4°C and the material was pooled
and aliquoted into Eppendorf tubes before longterm storage at -80°C. Sonicated material waschecked for sizing versus unsonicated control
after input DNA prep on either a fragment
analyzer or TapeStation.

Protocol Details
Bead Ruptor Homogenization:
Small pieces of previously fixed tissue weremoved to a 2 mL Omni Bead Ruptor tube
containing three 5 mm steel beads and 1 mLof Cell Shearing buffer (+PIC/PMSF) or Cell
Shearing Buffer without N-lauryl sarcosine (+PIC/PMSF). Kidney, PBMCs, liver, and brain were
homogenized at 4 m/s for 20 seconds Lung, heart,ovary, uterus, and adipose were homogenized 2for cycles of 4m/s for 20 seconds with ~2 minutes
between cycles.
After homogenization, samples homogenizedin Cell Shear without N-lauryl sarcosine werecentrifuged at full speed for 3 mins to pellet
material and resuspended in 1 mL of Cell Shearing
buffer (+PIC/PMSF).
PIXUL Sonication:
After Bead Ruptor homogenization, 100 µL/wellof homogenized suspension was moved into 1
column (8 wells) of a 96-well U-bottom plate andsonicated on PIXUL at a pulse of 50, PRF 1, burstof 20 for 72 mins (36 mins tested for liver andkidney only).

Figure 2: Overview of human tissues sonicatedwith PIXUL™ Multi-Sample Sonicator. All but one
sample surpassed our internal QC metric of 70%
sonicated fragments.
Kidney and adipose tissue nearly sonicate to100%. Some tissues, such as heart, are morechallenging, but this sample also approached
a 70% sonication level which is considered
acceptable for downstream ChIP-Seq analysis.

ChIP-Seq
After fragmentation, the samples were subjected
to ChIP-Seq analysis using 10 µg of sonicatedchromatin per reaction and 4 µg of an AbFlex ®
Histone H3K9ac (Cat. No. 91103) antibody, whichmarks active chromatin.

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TECHNOTE:PIXUL & CHIP-SEQ WITH LIMITED AMOUNTS OF TISSUE SAMPLES

Library preparation was carried out using theActive Motif Next Gen DNA Library Kit(Cat. No. 53216, Cat. No. 53264). The sequencing
was completed on the Illumina NextSeq500
and utilized approx. 20M reads for each of thesamples.

In contrast to that, no crosstalk is observed in anyof the other tissues that were surveyed, indicatinga strong tissue specificity from these experiments
using the PIXUL™ Multi-Sample Sonicator.

Pictured above, genome browser tracks of ChIPSeq results at the CD3 locus which specificallymarks T-cells. This is a sub-unit of the CD3
receptor complex where strong H3K9 acetylationis only observed throughout the entire gene
body in the PBMC population, as peripheral
blood mononuclear cells play a central role in theimmune response.

Here, we demonstrate the suitability of thePIXUL™ Multi-Sample Sonicator for preparation ofclinically relevant sample types and amounts. Withthe PIXUL™ Multi-Sample Sonicator it is possible
to sonicate chromatin under a standard set ofparameters, yielding material that is appropriately
sheared to the correct size and suitable fordownstream ChIP-Seq analysis.

Summary

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TECHNOTE:PIXUL & CHIP-SEQ WITH LIMITED AMOUNTS OF TISSUE SAMPLES
The main advantages of the PIXUL are:●

 igh throughput, tissue-specific results from PIXUL-sonicated chromatin to analyze tissue
H
specific differences in their biology under a uniform, time-saving set of parameters. The resulting
chromatin performs reliably in ChIP-Seq analysis.

●

 pigenomic profiling via ChIP-Seq was performed in 10 human tissues and 7 mouse tissues forE
3 histone modifications and 1 transcription factor by a single scientist in a few weeks (only thehuman tissues and 1 mark are shown in this Technote). This was previously impossible due to linear
processing of single samples, inconsistent shearing and cumbersome optimization procedures ofavailable sonicators.

●

 IXUL™ Multi-Sample Sonicator presents a new and robust technology for consistent andP
fast chromatin shearing for clinical volumes of samples, enabling epigenomic studies andtranslational research.

CHECK OUT OUR QUICK GUIDE:SHEARING CHROMATIN FROM FROZEN TISSUES WITH PIXULTM
Get recommendations for fragmenting chromatin with the PIXUL Multi-Sample Sonicator using
the PIXUL Chromatin Shearing Kit (Cat. No. 53132) using both human and mouse tissues.”

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