A P P L I C AT I O N 2 0 2 2 0 6

From Sample Prep to Sonication:
A ChIP-Seq Analysis Workflow
from Various Tissues Using theOMNI Bead Ruptor Elite™ Bead
Mill Homogenizer and PIXUL™
Multi-Sample Sonicator

Bead Ruptor Elite™
Bead Mill Homogenizer

OMNI International, a PerkinElmer company
Active Motif

Summary
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. Withsuch limitations from small amounts of starting material and difficult-to-process samples, consistency in dataand consistency in experiments is more important than ever. High quality tissue homogenization and consistentshearing is pivotal to generate data.
Herein, we demonstrate a robust workflow for ChIP-Seq sample preparation and analysis from clinically relevant
tissue types using the Bead Ruptor Elite™ Bead Mill Homogenizer to dissociate tissues and the PIXUL™ MultiSample Sonicator for ChIP-Seq sample preparation.

Materials and Methods
EQUIPMENTOMNI Equipment• OMNI Bead Ruptor Elite™ Bead Mill Homogenizer (Cat No. 19-042E)• 2 mL Reinforced Tubes with Screw Caps & Silicone O-Rings (Cat No. 19-648)• 5 mm Stainless Steel Beads Bulk, 325 g (Cat No. 27-7005)
For research use only. Not for use in diagnostic procedures.
Bead Ruptor Elite™
Bead Mill Homogenizer

Active Motif Equipment & Reagents• PIXUL™ Multi-Sample Sonicator (Cat No. 53130)• AbFlex® Histone H3K9ac Antibody (Cat. No. 91103)• Next Gen DNA Library Kit (Cat No. 53216, 53264)
For research use only. Not for use in diagnostic procedures.

PIXUL™
Multiple Sample Sonicator

Active Motif
1914 Palomar Oaks Way, Suite 150
Carlsbad, CA 92008(+1) 760-431-1263
www.activemotif.com

PROCEDURE
Fixation of Primary Tissues
Primary tissues were harvested 24 hours post-mortem and sent in preservation media to the research lab.
Immediately upon arrival, tissues were washed in ice-cold 1X phosphate-buffered saline (PBS) (Gibco, Cat No.
20012050), diced with a razor blade in PBS + 4 % formaldehyde, transferred to a conical tube and fixed for 15
minutes at room temperature with overhead rotation. The reaction was quenched with 1/20th volume 2.5 M
glycine for 5 minutes at room temperature with overhead rotation. Following fixation, samples were centrifuged
to remove the media before being snap-frozen in liquid nitrogen for long term storage at -80 °C.
Sample Preparation
Small pieces of previously fixed tissue were moved to a 2 mL reinforced tube (OMNI, Cat No. 19-648) containing
three 5 mm steel beads (OMNI, Cat No. 27-7005) and 1 mL of Cell Shearing Buffer containing reagents asrecommended by the ChIP protocol. Kidney, peripheral blood mononuclear cells (PBMCs), liver, and brain werehomogenized using the OMNI Bead Ruptor Elite™ Bead Mill Homogenizer (OMNI, Cat No. 19-042E) at 4 m/s for
20 seconds, while lung, heart, ovary, uterus and adipose were homogenized at 4 m/s for 2 cycles of 20 seconds
with a 2-minute dwell.
Following homogenization, those samples prepared in Cell Shearing Buffer containing recommended reagentswere centrifuged at 15,000 x g for 3 mins to pellet material before resuspension in 1 mL Cell Shearing Buffer
with added reagents.
PIXUL™ Multi-Sample Sonication
After homogenization on the Bead Ruptor Elite™ Bead Mill Homogenizer, 100 μL/well of homogenized suspension
were moved into 1 column (8 wells) of a 96-well round-bottom plate and sonicated on the PIXUL™ Multi-Sample
Sonicator (Active Motif, Cat No. 53130) 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 centrifuged at 15,000 x g for 1 min at 4 °C and the samples werepooled and aliquoted into Eppendorf tubes for long term storage at -80 °C. Sonicated material was comparedwith a non-sonicated control after input DNA prep on either Fragment Analyzer or TapeStation.
ChIP-Seq
After fragmentation, the samples were subjected to ChIP using 10 μg of sonicated chromatin per reaction and 4 μgof an AbFlex® Histone H3K9ac (Active Motif, Cat. No. 91103) antibody, which marks active chromatin. Less than 10μg of sonicated chromatin may be required depending on antibody. Other Active Motif AbFlex® antibodies used inthis experiment but not shown here are H3K27ac (Active Motif, Cat. No. 91193), H3K36me3 (Active Motif, Cat. No.
91265), and CTCF (Active Motif, Cat. No. 91285). The Low Cell ChIP-Seq Kit (Active Motif, Cat. No. 53084) was partof the protocol. Some other available options for consideration are PIXUL™ Chromatin Shearing Kit (Active Motif,Cat. No. 53132), PIXUL™ Chromatin Input Preparation Kit (Active Motif, Cat. No. 53134), etc.
Protein A/G agarose beads were prepared depending on the antibody. Chromatin was precleared.
Immunoprecipitation was performed on end-to-end rotor at 4⁰C overnight followed by washing, elution, reversalof cross-links and DNA purification.
For ChIP-Seq, library preparation was conducted using the Active Motif Next Gen DNA Library Kit (Cat. No.
53216, Cat. No. 53264). The sequencing was completed on the Illumina NextSeq500 and utilized approx. 20 M
reads for each of the samples.

RESULTS

Figure 1. PIXUL™ Multi-Sample Sonicator for Preparation of DNA from Brain Tissue. Lane 1 shows thenon-sonicated control, lane 2 shows the same sample sonicated for 72 minutes on PIXUL™ Multi-SampleSonicator. The sonicated sample shows an enrichment of fragments around 300 base pairs, which is theideal size for ChIP-Seq analysis.

Figure 2. Overview of human tissues sonicated with PIXUL™ Multi-Sample Sonicator.
All but one sample surpassed our internal QC metric of 70 % sonicated fragments.
Kidney and adipose tissue nearly sonicate to 100 %. Some tissues, such as heart, aremore challenging, but this sample also approached a 70 % sonication level which isconsidered acceptable for downstream ChIP-Seq analysis.

Figure 3. Genome browser tracks of ChIPSeq results for all tissue types at the CD3
locus which specifically marks 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, asPBMCs play a leading role in the immune
response. In contrast to that, no crosstalk
is observed in any of the other tissues thatwere surveyed, indicating a strong tissue
specificity from these experiments using
the PIXUL™ Multi-Sample Sonicator.

Conclusion
Herein, we demonstrate the suitability of the ChIP-Seq workflow using the Bead Ruptor Elite™ Bead Mill Homogenizerfor upstream tissue dissociation and PIXUL™ Multi-Sample Sonicator for preparation of relevant sample types andamounts. The Bead Ruptor Elite™ Bead Mill Homogenizer streamlines sample preparation allowing for quick tissue
dissociation, releasing analytes of interest for downstream analysis. Alongside the PIXUL™ Multi-Sample Sonicator,
it is possible to sonicate chromatin under a standard set of parameters and in a high-throughput manner, yielding
material that is appropriately sheared to the correct size and suitable for downstream ChIP-Seq analysis.
The PIXUL™ Multi-Sample Sonicator presents a new and robust technology for consistent and fast chromatin
shearing for clinical volumes of samples, enabling epigenomic studies and translational research.

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OMNI International, a PerkinElmer company
Representative:
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www.omni-inc.com

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07/20/2022