Enabling Epigenetics Research

TECHNICAL NOTE

Chromatin Sonication Recommendations for ChIP
SUMMARY

Chromatin immunoprecipitation (ChIP) is an assay used to identify sites in the genome wheretranscription factors bind or histones have undergone post-translational modifications. An
important step in the ChIP assay workflow is fragmentation of the chromatin, as fragment length
determines the resolution of site identification. This technical note describes points to consider
when optimizing sonication of your sample prior to performing ChIP.

FIXATIONSONICATION

Formaldehyde: Methanol-Buffered orMethanol-Free?
Formaldehyde is the most widely-used fixative to fix
chromatin for ChIP assays, and we at Active Motifhighly recommend it. Most sonicators are compatible
with methanol-buffered formaldehyde. However,some multi-sample sonicators require methanol-freeformaldehyde. Please see the table on the following
page for more details.

Variables that Affect Sonication
Many different factors can affect how well your sample
is sonicated: the sample type, amount, and volume,
crosslinking conditions, sonicator and tube type, andbuffer composition, to name a few. Therefore, it isimportant to re-optimize your sonication protocol if
any variables change.

Formaldehyde Alternatives & Additives
Active Motif’s ChIP kits have been optimized for
fixation with formaldehyde but there are somemodifications to the standard formaldehyde fixation
protocol that can provide advantages for certain
applications. For instance, when performing ChIP for
histone modifications, you can choose not to fix at all(native ChIP). For weak, transient, or indirect bindingof proteins to DNA, you may consider adding
another crosslinker to formaldehyde to extend thecrosslinking distance, such as dimethyl adipimidate(DMA), disuccinimidyl glutarate (DSG), or ethylene
glycolbis(succinimidyl succinate) (EGS).
The Dangers of Overfixing
Chromatin in overfixed samples is difficult tosolubilize, which can lead to poor results when usedin ChIP assays. Pay close attention to the sample
concentration, the final formaldehyde concentration,and the length of time your sample is fixed. Using 1%
formaldehyde for 10-15 minutes is the most common
fixation condition for cells, cell lines, and tissues.

Fragmentation Sizes
ChIP usually requires chromatin to be sheared to a sizeof 200-1200 bp, with a majority of ChIP applications
further limiting that range to 200-600 bp (e.g. ChIPSeq). It is important to optimize your shearing toproduce fragments of optimal size for your application.
Sonicate and Chill
Sonication heats samples, and overheating can cause
irreversible damage to the DNA or protein targets
which would significantly decrease the quality of yourChIP data. It is important to keep samples cold bysonicating on a chilled sonication platform or restingon ice between sonication cycles.
Enzymatic ShearingEnzymatic shearing is an option for researchers
without access to a physical shearing instrument or
performing native ChIP. However, enzymatic shearing
is plagued by bias, and may not shear chromatin fromdifficult cell lines, primary cells, or tissues. We at Active
Motif consider physical shearing with a sonicatorthe best practice, and only recommend enzymatic
shearing when sonication is not an option.

VERIFY FRAGMENT SIZE
After shearing, we recommend treating a smallaliquot of your sheared chromatin with RNase A andProteinase K, reversing the crosslinking, and purifying
the DNA to run on a 1.5% agarose gel for verificationof the sonication efficiency. DNA should appear asa smear anywhere between 200 – 1200 bp. In theimage to the right, Sample 1 omitted denaturing thesample with 500 mM NaCl (Step 14 of Section B in
the ChIP-IT High Sensitivity® Manual) and does notlook like well-sonicated chromatin. Sample 2 showsthe expected fragmentation between 200-1200 bp.

Agarose Gel of Sheared Chromatin. 500 ng of chromatin from
MCF-7 cells were fixed, sonicated, and run on a 1.5% agarosegel, according to the ChIP-IT High Sensitivity® Protocol.

WHERE TO BEGIN?
The table below lists recommendations for where to begin your sonication optimization. These parameters
come from the peer-reviewed publications referenced in the column on the right. Remember, these are juststarting points and it’s still a good idea to optimize for your specific conditions! For more information on
performing ChIP with Active Motif reagents, please visit our website.

Sonicator
Active Motif PIXUL™
Multi-Sample Sonicator

Fixative

Shearing Conditions

Methanol-buffered Pulse: 50 2 MHz cycles/pulse
or methanol-free Pulse Repetition Frequency: 1 kHz
formaldehyde
Process Time: 36 minutes for 1-96 samples
Burst Rate: 20 Hz

References (PMIDs)
30927002

NOTE: We recommend diluting thesheared chromatin 5-fold in Active Motif
ChIP buffer prior to performing ChIP.
Active Motif EpiShear™
Probe Sonicator
Covaris® FocusedUltrasonicator
Models:M220, E220, S2, S220

Methanol-buffered Amplitude: 25%
formaldehyde
Pulse: 30 seconds ON, 30 seconds OFFCycles: 20
Methanol-freeformaldehyde

Shearing Buffer: Buffer D3
Peak Power: 75 watts
Duty Factor: 10.0
Cycles/Burst: 200Time: 600 sec

27460500
28455377
28399410
24466341
29093577
28729413

NOTE: We recommend diluting thesheared chromatin 5-fold in Active Motif
ChIP buffer prior to performing ChIP.
Diagenode Bioruptor®
Models:Pico, Plus, UCD-200

Methanol-buffered High Poweror methanol-free Pulse: 30 seconds ON, 30 seconds OFF
formaldehyde
Cycles: 7-12

29317594
28030801
25241747
30139998

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