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Optimizing a Dual Fixation Protocol to Study Protein
Complexes Binding to Chromatin in vivo
Benjamin Carter 1, Emily Dykhuizen 1, Sean Gerrin 2, and Hamid Khoja 2
Author Affiliation:
1. Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, IN 47907-2063 USA
2. Covaris, Inc., Woburn, Massachusetts 01801-1721 USA

Introduction

Materials and Methods

Chromatin immunoprecipitation coupled with next-generation

Instrument and Consumables

sequencing technology (ChIP-seq) has enabled investigators• E220 Focused-ultrasonicator (Covaris, PN 500239)

to generate high-resolution maps of genomic binding sites for• Rack 24 Place milliTUBE 1 mL (Covaris, PN 500368)

transcription factors and other members of the chromatin-• milliTUBE-1mL with AFA fiber (Covaris, PN 520130)
Reagents

associated machinery.

• truChIP Chromatin Shearing Kit (Covaris, PN 520154)
Many epigenetic factors that associate with chromatin do so as• DMA (Thermo Fisher, PN 20660)

components of large multi-subunit complexes in which only a• DSG (CovaChem, PN 13301)

subset of the members directly bind to chromatin . For example,• EGS (CovaChem, PN 13308)

1members the mammalian SWI/SNF chromatin remodeling complex
BAF, which promotes proper gene expression and chromatin

Dual Fixation Protocol

dynamics, commonly associate with chromatin in assemblies ofApproximately 30 million Caki2 kidney clear cell carcinoma cells inover ten subunits

15 cm plates were washed once with 5 mL of ice-cold 1X PBS. This

2,3. The use of ChIP-seq to map the genomic

binding of specific members of such complexes often requireswash was aspirated, and the cells were rocked in a 5 mL solution

stabilization of the complex via protein-protein crosslinkingof 3 mM EGS, DSG, and DMA crosslinkers in 1X PBS for 5 min attreatments.

room temperature. This solution was aspirated, and the cells were
washed twice with ice-cold 1X PBS. A second crosslinking step

ChIP requires optimization of fixing conditions for each tissue or

was performed by rocking the cells in 5 mL of 1% formaldehydecell line and for each protein complex being studied. Importantly,in 1X Fixing Buffer A at room temperature for 1 min. 300 µL ofoptimization is required for the subsequent shearing of theQuenching Buffer E was added, and the cells were rocked for anchromatin to obtain fragments within the distribution size required

additional 5 min. The cells were scraped from the 15 cm plates

for sequencing while simultaneously preserving the integrity of theusing a plastic scraper and two 5 mL 1X PBS washes and wereepitope.

transferred to a 15 mL conical vial. The cells were then pelletedby room temperature centrifugation for 5 min at 200 x g. Two

Here, we tested a Covaris-developed chromatin sample

washes were performed by suspending the cells in 5 mL of ice-cold

preparation protocol to effectively study the interactions of protein

1X PBS followed by centrifugation. The final wash was aspirated

complexes associated with chromatin. An initial crosslinking

completely, and the cells were placed on ice before proceeding tococktail was used prior to carrying out the Covaris truChIP ®

nuclei preparation.

Chromatin Shearing Protocol. Relative to a standard single fixation
method, this Covaris-developed protocol enhanced signal overSingle Fixation Protocol

background at known binding sites for members of the BAF

A standard single-fixation protocol was performed as acomplex including the ATPase subunit, BRG1 . In this applicationcomparative control for the dual fixation method described above.

note, we provide shearing profiles, evaluate epitope integrity

After collection and washing of the Caki2 cells, crosslinking was

at each shearing time, and ChIP-qPCR results obtained using

performed by rocking the cells in 5 mL of 1% formaldehyde in

the single step formaldehyde fixation and the dual crosslinking

1X Fixing Buffer A at room temperature for 5 min. 300 µL ofprotocol.

Quenching Buffer E was added, and the cells were rocked for an41

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additional 5 min. The cells were scraped from the 15 cm plates

Evaluation of Epitope Integrity

using a plastic scraper and two 5 mL PBS washes and were

Epitope integrity was assayed via Western blot. In short, 10 µL

transferred to a 15 mL conical vial. The cells were then pelletedof each sheared chromatin sample was combined with an equalby room temperature centrifugation for 5 min at 200 x g. Twovolume of 2X Laemmli buffer (4% SDS, 20% glycerol, 120 mM

washes of were performed by suspending the cells in 5 mL ofTris pH 6.8, bromophenol blue) and heated at 60C for 10 min on

ice-cold 1X PBS followed by centrifugation. The final wash wasa thermal cycler. The samples were then loaded on a NuPAGE

aspirated completely, and the cells were placed on ice before

precast gradient gel (Thermo Fisher Scientific) and run at aproceeding to nuclei preparation.

constant current of 40 mA per gel for 1h 15 min. Proteins weretransferred to an Immobilon-FL membrane in Towbin buffer (25Nuclei PreparationmM Tris, 129 mM glycine, 10% methanol, pH adjusted to 8.3) at

Cell lysis and isolation of nuclei were performed as directed in the120 V for 1h. Western blotting was performed by blocking theCovaris truChIP protocol. In short, cells were suspended in 1 mL

membrane with 5% BSA for 1h, incubating with a 1:10,000 dilutionof Lysis Buffer B, and the suspension was transferred to a 1.5 mLof the primary antibody overnight at 4C, washing the membranemicrocentrifuge tube. The sample was rotated at 4C for 10 min

3X with PBS-T, and rocking the membrane in a 1:10,000 dilution offollowed by centrifugation at 1,700 x g for 5 min. The lysis buffer

LI-COR IRDye secondary antibody for 1h. Bands were visualized

was discarded, and the cell pellet was suspended in 1 mL of Wash

using a LI-COR Odyssey fluorescence imager.

Buffer C followed by an additional rotation and centrifugation
with the same parameters. The wash buffer was removed, andResults and Discussion

the inner surfaces of the tube were washed with 1 mL of Shearing

Results

Buffer D3. The sample was centrifuged, and the shearing buffer

Caki2 renal carcinoma cells were harvested and fixed using either

was discarded. This wash step was repeated one additional time.

the dual crosslinking method or the standard formaldehyde-onlymethod (Figure 1). Cell lysis, nuclear isolation, and chromatin

Chromatin Shearing

purification steps were performed according to the truChIP

The supernatant was aspirated completely and the nuclear pellet

Chromatin Shearing Protocol. Electropherogram trace files werewas suspended in 1 mL of 1X Shearing Buffer D3. This suspension

generated from the shearing time course performed (Figure 2).

was transferred to a prechilled Covaris milliTUBE 1ml AFA Fiber

Input samples were prepared, and DNA fragment size distributions(PN 520130). Shearing was performed using a Covaris E220

were calculated for over sheared (41 to 150 bp) optimally sheared

Focused-ultrasonicator with the recommended settings: 140 PIP,

(151 to 700 bp), and under sheared (701 to 7,000 bp) size ranges

5% duty factor, and 200 CPB. A time course was performed using(Table 1).

3, 6, and, 9 minutes as the pre-defined processing intervals. 60 µLof sample was recovered at each time point for sizing and epitope
integrity analysis. 60 µL of Shearing Buffer D3 was added toreplace the volume removed at each time point.
Chromatin Fragment Sizing

Dual Fixation Chromatin
Sample Preparation Workflow

Single Fixation Chromatin
Sample Preparation Workflow

Culture Cells as a Suspension
or Adherent Monolayer

Culture Cells as a Suspension
or Adherent Monolayer

Protein-protein Fixation Cocktail(3mM, DSG, EGS, DMA)Fix Cells with 1% Formaldehyde

Wash Twice with 1X PBS

Nuclei Preparation

Fix Cells with 1% Formaldehyde

Chromatin Shearing withCovaris AFA

Chromatin shearing was analyzed by preparing an input sample
and determining the size distribution for the chromatin fragments
using an Agilent bioanalyzer. To prepare the input samples, 1µL of 10 mg/mL RNAse A was added to 25 µL of each shearedchromatin sample. The samples were incubated at 37C for 30 min
to degrade the RNA. 1 µL of 10 mg/mL proteinase K was thenNuclei Preparation

added to the samples followed by an overnight incubation at 65C
on a thermal cycler to reverse the crosslinking. The next day, theChromatin Shearing withCovaris AFA®

DNA was purified using a QIAquick PCR Purification Kit (Qiagen).

Figure 1. Comparison of dual-fixation and single-fixation workflows.

DNA fragment size distributions were then determined for theresulting purified input DNA samples using the DNA 12000 chip
(Agilent Technologies).

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Shearing Time Course Evaluation
A.

3 Minutes

D.

3 Minutes

B.

6 Minutes

E.

6 Minutes

C.

9 Minutes

F.

9 Minutes

Figure 2. A-C: electropherogram trace files for samples processed using the dual fixation method and sheared for the indicated time. D-F: electropherogram trace files for samples
processed using the single fixation method and sheared for the indicated time.

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Formaldehyde Only

6Dual Fixation

3 min

6 min

9 min

3 min

6 min

9 min

41 to 150 bp

6%

9%

12%

3%

7%

11%

151 to 700 bp

79%

83%

81%

54%

72%

78%

701 to 7,000 bp

13%

5%

3%

41%

18%

9%

Fold Enrichment over IgG

Fragment Size Range

Table 1. Size distribution of chromatin fragments. Using the regional table functionin the Bioanalyzer software, the percentage of fragments within the ranges werecalculated for the two fixation methods defined.

321GRID1TXNRD1

Dual Fixation

EEF1E1

Mock (IgG)

Formaldehyde OnlyFigure 3. ChIP-qPCR signal over background at three enriched genes and mocks (IgG).
ChIP-qPCR analysis of BRG1 enrichment at three promoter regions enriched for theBAF complex in Caki2 cells. Mock immunoprecipitations (IP) are plotted for both thesingle and dual fixation methods to assess the background non-specific binding. Foldenrichment is displayed relative to the IgG mock ChIP treatment. Error bars indicate
one standard deviation between two biological replicates.

good fragment size distributions at 6 and 9 minutes of shearing. To
evaluate epitope integrity under these conditions, we performed
immunoblots of BRG1, a large BAF complex subunit that migrates
over 250 kDa, using an anti-BRG1 primary antibody. We observedthat epitope integrity was maintained through 6 minutes of shearing

Discussion

in both samples, and through 9 minutes of shearing with the dual

In this study, we tested the dual fixation protocol for ChIP-qPCR

fixation method (Figure 2).

and ChIP-Seq analysis of BAF complex subunits in Caki2 cells.

Processing Time (min)Formaldehyde Only
640

Both the dual fixation and formaldehyde-only methods yielded

35Whereas formaldehyde is typically used to study interactions inDual Fixation
936very close proximity, the chemical crosslinking agents used in the9dual fixation protocol exhibit a variety of crosslinking distances. Thisenhances stabilization of protein complexes relative to the singlefixation method. Examination of chromatin shearing profile of dual
fixed samples using Agilent bioanalyzer indicated improvement

Figure 2. BRG1 (250 kDa) epitope integrity after shearing. Western blot results
demonstrate that epitope integrity was maintained through 6 minutes of shearing inboth samples, and through 9 minutes with the dual fixation method.

in fragment size distribution as compared to formaldehyde only
fixed samples. Western blot analysis of the sheared chromatinalso indicated greater epitope integrity for dual fixed samples

Based on the DNA fragment size distributions and the epitope

when probed with the same antibody used for ChIP. Compared tointegrity blot, we proceeded with a 6 minute shearing time fora standard formaldehyde-only fixation method, the dual fixation

the formaldehyde-only method and a 9 minute shearing time for

protocol enhanced signal over background up to twofold for ChIP-

the dual fixation method. Using these parameters, we performed

qPCR of BRG1. Taken together, the results indicate that the dual

ChIP-qPCR using an anti-BRG1 antibody and primers that amplified

fixation method improves the detection of chromatin-associatedputative BRG1 peaks based on ChIP-seq datasets from similar cellmachinery in ChIP assays. With optimization of fixation andlines. We found that the dual fixation method resulted in increased

shearing times, this protocol is suitable for use in other cells lines

enrichment over background for the three peak regions relative toand conditions.

the standard single fixation method (Figure 3).

References
1.				

Tian B, Yang J, Brasier AR: Two-step cross-linking for analysis of protein-chromatininteractions. Methods Mol Biol 2012, 809:105-120.
DOI: 10.1007/978-1-61779-376-9_7

3.				

Sokpor G, Xie Y, Rosenbusch J, Tuoc T: Chromatin Remodeling BAF (SWI/SNF)
Complexes in Neural Development and Disorders. Front Mol Neurosci 2017, 10:243.
DOI: 10.3389/fnmol.2017.00243

2.				

Pulice JL, Kadoch C: Composition and Function of Mammalian SWI/SNF Chromatin
Remodeling Complexes in Human Disease. Cold Spring Harb Symp Quant Biol 2016,
81:53-60. DOI: 10.1101/sqb.2016.81.031021

4.				

Savas S, Skardasi G: The SWI/SNF complex subunit genes: Their functions, variations,and links to risk and survival outcomes in human cancers. Crit Rev Oncol Hematol 2018,
123:114-131. DOI: 10.1016/j.critrevonc.2018.01.009

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M020090_RevB_Feb2019 | Information subject to change without notice | For research only | Not for use in diagnostic procedures | 2019© Covaris, Inc.

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