ChIP-IT® FFPE
Chromatin Preparation II(version A1)
Catalog No. 53031

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Information in this manual is subject to change without notice and does not constitute a commitment on the part of Active Motif, Inc. It is supplied on an “as is” basis without any warranty of any
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TABLE OF CONTENTS

Page

Overview  . . . . . . .  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Flow Chart of Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Introduction  . . . .  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3
References . . . . . .  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3
Kit Performance and Benefits  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
ChIP-IT FFPE Chromatin Preparation II . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Kit Components and Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Additional Materials Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Protocol Overview and Time Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Protocols – Experimental Set Up
Buffer Preparation  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Protocols – Preparation of Sheared Chromatin
Section A. Removal of Paraffin & Rehydration of FFPE Tissue Slides . . . . . . . . . . . . . . . .  10
Section B. Removal of Paraffin & Rehydration of FFPE Tissue Blocks . . . . . . . . . . . . . . . . . 11
Section C. Tissue Homogenization and Chromatin Isolation . . . . . . . . . . . . . . . . . . . . . . . . 12
Section D. Input Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Section E. Analysis of Input DNA and Chromatin Quality . . . . . . . . . . . . . . . . . . . . . . . . .  14
Section F. Additional Sonication of Insoluble Pellet . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  16
Appendix
Section G. qPCR Primer Design and Data Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Section H. Troubleshooting Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  18
Section I. Related Products  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  19
Technical Services  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

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Overview
Chromatin Immunoprecipitation (ChIP) is a powerful tool for studying protein/DNA interactions,
including transcription factors, co-regulatory proteins, modified histones, chromatin-modifying
enzymes and polymerases because it enables identification of the localization of proteins boundto specific DNA loci. When used in combination with whole-genome analysis such as ChIP-Seq,
insights are possible into gene regulation, gene expression, mechanisms of chromatin modificationand pathway analysis.
Formalin-fixed paraffin-embedded (FFPE) tissue blocks and histology slides are a valuable resource
for retrospective research on clinical samples. Clinical information, treatments and outcomes areoften available for these sample types and large collections of FFPE material is commercially available. The ability to study FFPE samples provides researchers with an opportunity to link FFPE data
to disease, diagnosis and biomarker discovery. Traditionally, FFPE samples have not been useful inchromatin immunoprecipitation because of the limited size of the samples, and the fact that theformalin fixation process often causes degradation and loss of antigenicity.
Active Motif’s ChIP-IT® FFPE Chromatin Preparation II and ChIP-IT® FFPE II Kits are our second
generation FFPE ChIP kits in which the cumbersome de-paraffinization and dehydration procedure
is streamlined to allow the preparation of high quality ChIP-enriched DNA using less reagents andhands on time. This streamlined chromatin preparation protocol is optimal when working withlimited amounts FFPE tissue. The ChIP-IT FFPE II Kit has been optimized using specially formulated
reagents and protocol guidelines to increase sensitivity and enable analysis by both qPCR and
Next Generation sequencing from extremely limited starting material, while producing minimal
background signal, thereby enabling specific detection of the target protein of interest.
The ChIP-IT FFPE Chromatin Preparation II Kit is designed for use with high quality FFPE samples(low quality samples should be prepared using the first generation ChIP-IT FFPE Chromatin Preparation Kit). This kit contains sufficient reagents for 5 chromatin preparations using up to 5 unstained
tissue slides or FFPE block sections per chromatin preparation. It is necessary to use Active Motif’s
ChIP-IT FFPE II Kit for the downstream ChIP reactions due to the limited yields obtained fromFFPE samples. The ChIP-IT® qPCR Analysis Kit (Catalog No. 53029) can be used following ChIP to
examine site-specific ChIP enrichment. For the preparation of Next generation sequencing (NGS)
libraries from the ChIP-enriched DNA we recommend that you use the Next Gen DNA Library
Kit which is 500-fold more sensitive than other library preparation kits. To learn about available
ChIP-IT® Control Kits, control qPCR primer sets, ChIP-Seq validated antibodies, or Active Motif’s
EpiShear™ sonication devices, please visit our website at www.activemotif.com/chip.
product

formatcatalog no.

ChIP-IT® FFPE Chromatin Preparation II Kit

5 rxns53031

ChIP-IT® FFPE II

16 rxns

53047

Next Gen DNA Library Kit

16 rxns

53216

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1Flow Chart of Process

Flow Chart of the ChIP-IT FFPE Chromatin Preparation II Kit.
In ChIP-IT FFPE Chromatin Preparation II, 10 µm sections from FFPE tissue blocks, or 5-10 µm sections from unstained FFPE
slides are used as the starting material. Three to five sections or slides can be used for each chromatin preparation in orderto obtain sufficient yield for downstream ChIP analysis. The paraffin is removed and the tissue is rehydrated from eachslide/tube. The tissue then undergoes sonication, reversal of crosslinks and purification. The soluble and insoluble chromatin is analyzed and quantified by qPCR, or alternatively, by fluorometer.

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2

Introduction
Formalin-fixed paraffin embedded (FFPE) samples represent an opportunity for researchers to
study clinical outcomes of disease and/or treatment conditions in the search to better understandthe disease, or as a mechanism to identify biomarkers for screening purposes. FFPE samples serveas the “gold standard” for pathology sample preservation and large collections of these tissues areavailable.
There are many challenges associated with working with FFPE samples. The samples are often
limited in size and require the use of multiple histological slides or tissue sections to extract sufficient quantities of material for downstream analysis. Another challenge is the lack of consistency
in the methodologies used for formalin fixation. Some treatments tend to be harsh, causing
degradation of the sample, loss of antigenicity, or they create “overfixed” chromatin which isdifficult to efficiently shear. Although FFPE samples have been used for high-throughput DNA andRNA analysis1,2, the challenges explained above have prevented FFPE material from being used inchromatin immunoprecipitation (ChIP).
ChIP itself can be technically demanding. ChIP requires high-quality antibodies to recognize thefixed, target-bound proteins of interest, and an efficient means to precipitate the antibody/chromatin complex (usually protein A or G beads). In addition, specialized buffers, inhibitor cocktails
and blocking reagents are required to minimize non-specific enrichment and reduce protein
degradation.
Researchers have started to address the need for a methodology to study the influences of epigenetics on normal and tumor samples beyond the traditional immunohistochemistry (IHC) analysis. Pathology tissue chromatin immunoprecipitation (PAT-ChIP) was the first method to extract
and analyze FFPE chromatin for use in high-throughput analysis, such as ChIP-Seq3,4. Subsequently,
many laboratories have attempted to prepare high quality chromatin from FFPE samples5, however
many are still reporting issues in generating quality chromatin from ChIP-seq analysis.
Active Motif has utilized our expertise with ChIP to develop a suite of kits for performing ChIP on
FFPE samples for use in Next-generation sequencing. The ChIP-IT FFPE Chromatin Preparation II Kit
contains specially formulated reagents and protocol guidelines to extract high quality chromatinfrom limited amounts of histological slides or tissue sections. Coupled with our newly developed
and most sensitive ChIP kit, ChIP-IT FFPE II, you have everything you need to generate high quality
ChIP-Seq data from extremely limited starting material while producing minimal backgroundsignal, thereby enabling specific detection of the target protein of interest. All Active Motif FFPEChIP kits contain controls to help validate results at each step of the process.

References
1.
2.
3.
4.
5.

Weng, L. et al. (2010) J Pathol., 222: 41-51.
Gu, H.., et al. (2010) Nat. Methods, 7: 133-136.
Fanelli, M. et al. (2010) PNAS, 107(50): 21535-21540.
Fanelli, M. et al. (2011) Nat. Protocols, 6(12): 1905-1919.
Cejas, P. et al. (2016) Nat. Med., 22(6): 685-91.
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3Kit Performance and Benefits
ChIP-IT FFPE Chromatin Preparation II Advantages:• Easily obtain chromatin using limited amounts of histological slides or tissue blocks as thestarting material• Optimized reagents help preserve the quality of the chromatin during extraction• Highly robust procedure has been validated using FFPE chromatin from both normal andtumor samples with proven performance in both qPCR and ChIP-Seq analysis• Includes positive control DNA and PCR primers to quantify the chromatin and confirm theshearing efficiency prior to use in ChIP

Detection limit: The limits of the assay will depend on the size and tissue type and may require
optimization. The ChIP-IT FFPE Chromatin Preparation II kit was specifically developed for extracting high quality chromatin for use in ChIP from limited, but high quality, FFPE tissue slides orblocks. The protocol was optimized to require only five 5 µm sections per chromatin preparation. Our protocol offers guidelines and troubleshooting tips for processing samples to obtain therequired 200 ng minimum of chromatin per ChIP reaction. Chromatin should be validated prior touse in the ChIP-IT FFPE II Kit (Catalog No. 53047).

Product Performance: The ChIP-IT FFPE Chromatin Preparation Kit assumes that FFPE tissues are
already available as standard sections. Due to the variability that exists in the formalin fixation
process and the storage conditions of the sample, not all FFPE material may yield high quality
chromatin. The ChIP-IT FFPE Chromatin Preparation II Kit provides recommendations and guidelines to assist in the extraction process, but Active Motif cannot guarantee successful chromatin
preparations for variables that fall outside of our assay kit.
If preparing samples for FFPE preservation, it is important to minimize the time between theresection of the tissue and the fixation in formalin to generate high quality FFPE tissues. Samples
should be fixed immediately and stored in a cool, dry location. With human samples, due to theneed for surgical removal, there is often variability in the time from resection to fixation. For
animal models, in vivo perfusion can be used to generate high quality FFPE tissues.

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4ChIP-IT® FFPE Chromatin Preparation II
Evaluating chromatin quantity and quality using qPCR

Figure 1. Analysis of chromatin quality and quantity using qPCR.
Samples of glioblastoma patient-derived xenografts were processed using the ChIP-IT FFPE Chromatin Preparation II Kit.
Three 5 µm curls were used for Sample GBM1, and cell pellets from dissociated tumor were used for Samples GBM2 and
GBM3. Samples were reverse cross-linked, proteinase K treated and purified for analysis by qPCR using the included DNA
standards and qPCR primer sets to determine the quantity and quality of the extracted chromatin. If sufficient quantities
are not obtained after a single sonication, additional rounds of sonication can be performed (as specified in Section F).

ChIP-IT FFPE ChIP-Seq Results

Figure 2: H3K27ac and H3K27me3 ChIP-Seq on Glioblastoma samples.
Samples of glioblastoma patient-derived xenografts were used in H3K27ac and H3K27me3 ChIP-Seq to generate genomewide profiles of this histone modification. Libraries were constructed using the Next Gen DNA Library Kit (Catalog No.
53216). A portion of that data is presented above.

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5Kit Components and Storage
Please store each component at the temperature indicated in the table below. Reagents are forresearch use only.
Reagents

Quantity

Storage

FFPE ChIP Buffer

2.5 mlRT

5M NaCl

125 µlRTTE, pH 8.0

2 x 1 ml

RT

FFPE Low EDTA TE

1 ml

RT

RNase A (10 µg/µl)

2 x 25 µl-20°C

Proteinase K (10 µg/µl)

50 µl

-20°C

Protease Inhibitor Cocktail

50 µl

-20°C

Deacetylase Inhibitor

80 µl

-20°C

Carrier2 x 25 µl

-20°C

PMSF

450 µl-20°C

DNA Standard AM1

20 µl

-20°C

DNA Standard AM2

20 µl

-20°C

DNA Standard AM3

20 µl

-20°C

Human Positive Control Primer Set GAPDH-2

350 µl-20°C

Additional materials required• Xylene• Microcentrifuge (table top centrifuge 4°C) and microcentrifuge tubes• 100% ethanol (absolute)• Nuclease- and protease-free water, such as Milli-Q H2O• Vortex• Heat block• Apparatus to rotate tubes end-to-end at 4°C (e.g. a Labquake from Barnstead/Thermolyne
with a tube holder for 1.5 ml microcentrifuge tubes)• Razor Blades• 250 µl PCR tubes• Thermocycler• Thermomixer• 15 ml conical tubes
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6• Phenol and chloroform/isoamyl alcohol (24:1) or Phenol/chloroform/isoamyl alcohol (25:24:1)(DNA Purification, Molecular Biology Grade)• Orbital Shaker• Fluorometer and/or qPCR instrument for DNA quantitation• Pipettors and tips (filter tips are recommended)• Sonicator (e.g. Active Motif’s EpiShear™ Sonicator with a 1/8” probe (Catalog No. 53051) with
the EpiShear™ Cooled Sonication Platform (Catalog No. 53080))• (Optional) Coplin jars
• (Optional) SYBR Green qPCR master mix (Bio-Rad Cat # 170-8882)

Protocol Overview and Time Table
Required Time
Deparaffinization and rehydration of FFPE material

1.5 - 2 hours

Tissue homogenization

1 hourSonication

1 hour per sample

Input Preparation

15 hours

qPCR Analysis of Input

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7

Protocols – Experimental Set Up
PLEASE READ THE ENTIRE PROTOCOL BEFORE STARTING!
Buffer Preparation
FFPE ChIP Buffer
Is provided ready to use.
Deacetylase Inhibitor
Thaw on ice.
Protease Inhibitor Cocktail (PIC)
Thaw the PIC at room temperature until fully dissolved, which takes about 30 minutes. Vortex
gently and spin down briefly before use, then add to the buffers immediately before use.
DNA Standards AM1, AM2 and AM3
Due to the low quantities of chromatin recovered from FFPE samples, it is necessary to quantify
the chromatin using qPCR analysis against known quantities of DNA, such as those provided in
DNA Standards AM1, AM2 and AM3. In the qPCR instrument, set the values for DNA Standard AM1
to 2.5 ng/µl, DNA Standard AM2 to 0.25 ng/µl and DNA Standard AM3 to 0.025 ng/µl. Use 2 µl ofeach DNA standard in the qPCR reactions.
Alternatively, a fluorometric method, such as Qubit™ Fluorometric Quantitation, can be used toquantify the chromatin input. Nanodrop readings are not sensitive enough to detect the lowquantities of DNA and, therefore, use of the Nanodrop for DNA quantification is not recommended.
Human Positive Control Primer Set GAPDH-2
The Human Positive Control Primer Set GAPDH-2 is provided for use in the qPCR quantification of
the chromatin input. This primer set can be used to amplify DNA Standards AM1, AM2, AM3 andchromatin input samples. The primer is provided at 2.5 µM. Use 1 µl per qPCR reaction. Alternatively, a positive control primer set specific for a human gene-of-interest can be used to amplify
the DNA Standards and chromatin samples for quantification.
If not using human material, you will need to design appropriate qPCR primer sets to match yourspecies. We recommend designing primers to perform at an annealing temperature of 58°C so thatall qPCR reactions can be performed under identical conditions. An amplicon length of 75-150 bp
is recommended. To see a list of validated species-specific qPCR primers designed according tothese recommendations, visit www.activemotif.com/chipprimers.

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8

Recommendations
Chromatin Shearing Tips
ChIP experiments require chromatin that has been sheared to a size of 200-1200 bp. Due to thelimited sample size and yield, there is usually insufficient chromatin available for analysis of shearing
efficiency by agarose gel electrophoresis. Instead, we suggest analyzing the quantity and quality of
the chromatin preparation through qPCR. The included DNA Standards and qPCR Primer set enables
quantification of the chromatin input. Both the soluble chromatin fraction and the insoluble pellet
can be analyzed to determine the efficiency of the chromatin preparation. If a majority of thechromatin is still found in the insoluble fraction, the pellet should undergo additional sonication andextraction preparation. The soluble chromatin that is obtained from the additional processing can be
combined with the original soluble fraction and quantified.
In general, shearing efficiency is improved through the use of a small shearing volume and a V-bottom tube rather than a round-bottom tube. Also, note that shearing is inefficient if the chromatin
sample becomes emulsified with air bubbles. To determine the appropriate shearing level for yoursample, set up a “practice” tube containing only ChIP Buffer. Slowly increase the sonication amplitude until foaming starts to occur. Reduce the amplitude setting down slightly and mark this as thehighest possible intensity to use without foaming. If a chromatin preparation becomes emulsified
inadvertently, discontinue shearing and centrifuge the sample at maximum speed for 4 minutes at
4ºC in a microcentrifuge to remove trapped air. Finally, to prevent overheating and denaturation ofchromatin, samples should be kept on ice as much as possible during shearing, and shearing shouldbe performed discontinuously (i.e. sonicate for 30 seconds, then place on ice/water for 30 seconds,
sonicate again for 30 seconds, etc.). If possible, shear while on ice or use Active Motif’s EpiShear
Cooled Sonication Platform (Catalog No. 53080) to help regulate sample temperature.
Chromatin Quantity
A minimum of 200 ng chromatin is needed per ChIP reaction in Active Motif’s ChIP-IT FFPE II Kit.
The volume of the ChIP reaction should not exceed 200 µl. If more chromatin is available, it isrecommended to use larger quantities per ChIP in order to improve the efficiency of the ChIP
reaction. If insufficient chromatin is obtained, we suggest evaluating the insoluble chromatin pellet
or processing additional tissue sections or slides to obtain the required chromatin quantity.
Safety Precautions
Xylene is a skin irritant. Appropriate safety precautions (i.e. safety glasses, gloves and lab coat)should be used. It is recommended to work with the xylene in a biosafety hood to avoid inhalation. Please discard the xylene into a glass container for organic solvents and dispose of in accordance with local and federal regulations.
Do not discard down the sink. Also, chromatin sonication should be performed in a biosafety
hood if the chromatin is extracted from biohazardous or infectious materials.

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9

Protocols – Preparation of Sheared Chromatin
Section A: Removal of Paraffin and Rehydration of FFPE Tissue
Sections Mounted on Slides
This protocol describes processing FFPE tissue sections mounted on slides. Depending on the size
and tissue type, it may be necessary to process multiple slides together within a single chromatin
preparation in order to obtain enough chromatin for DNA quantification and downstream ChIP
analysis using the ChIP-IT FFPE II Kit (Catalog No. 53047). This protocol is designed for a maximumof 5 slides to be combined per chromatin preparation. The kit contains enough material for 5
chromatin preparations and the analysis of 25 Input samples.
1.

Prepare appropriate number of slides (5-10 µm thickness) required to obtain sufficientsoluble chromatin for DNA quantification and ChIP analysis. A minimum of 200 ng chromatin
is required for each ChIP reaction in the ChIP-IT FFPE II Kit. We recommend to pool 3-5 slides
per chromatin preparation.

2.

Start the deparaffinization process in a fume hood by applying xylene to each slide to beprocessed so that the entire section is immersed in xylene. 1 ml xylene is sufficient for
approximately three slides. Loosely cover the slides (e.g. use the lid of a pipette tip box).
Incubate for 10 minutes at room temperature.

3.

Carefully discard the solution using the appropriate glass disposal container. Do not discard
down the sink. Dab the edge of each slide with a paper towel. Repeat Step 2 two more times
for a total of 3 treatments.

4.

An alternative to the deparaffinization procedure above is to fill a Coplin Jar with xylene(approximately 30-35 ml) and incubate the slides in the jar for 30 minutes.

5.

Transfer the deparaffinized tissue into a Coplin Jar containing absolute (100%) ethanol andincubate 10 minutes at room temperature. Alternatively, if not using Coplin Jars use a plastic
microscope slide box for the rehydration procedure.

6.

To start rehydration of the tissue, transfer the samples into a Coplin Jar containing 70% (vol/vol) ethanol solution and incubate 10 minutes at room temperature.

7.

Continue the rehydration process of Step 6 by progressively increasing the percentage ofwater to obtain 50% and 20% ethanol (vol/vol) solutions, respectively, for each wash step.
The final rehydration incubation should be in Milli-Q water only.

8.

Between the rehydration steps above prepare the appropriate number of 1.5 ml microcentrifuge tubes with 500 µl FFPE ChIP Buffer

9.

Carefully transfer the rehydrated tissue sections to a 1.5 ml microcentrifuge tube with 500 µl
FFPE ChIP Buffer containing 5 µl PIC and 5 µl PMSF by scraping the tissue off the slides usinga razor blade. If using multiple slides, you will want to combine the sample material into one
tube at this time.

10. Proceed immediately to Section C: Tissue Homogenization and Chromatin Isolation.

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10Section B: Removal of Paraffin and Rehydration of FFPE Tissue
Sections From FFPE Blocks
This protocol describes processing FFPE tissue sections from FFPE blocks. Depending on the size
and tissue type, it may be necessary to process multiple sections together within a single chromatin preparation in order to obtain enough chromatin for DNA quantification and downstream ChIP
analysis using the ChIP-IT FFPE II Kit (Catalog No. 53047). This protocol is designed for a maximumof five 10 µm sections to be combined per chromatin preparation. The kit contains enough material for 5 chromatin preparations and the analysis of 25 Input samples.
1.

Prepare appropriate number of tissue blocks (5-10 µm thickness) required to obtain sufficientsoluble chromatin for DNA quantification and ChIP analysis. A minimum of 200 ng chromatin
is required for each ChIP reaction in the ChIP-IT FFPE II Kit. Place 1-5 tissue sections into a 1.5
ml microcentrifuge tube. The number of sections per tube will depend on the size of thesample and how much tissue material will fit into a single tube.

2.

Working in a fume hood, start the deparaffinization process by applying 1 ml xylene to eachtube. Incubate for 10 minutes at room temperature on a rotating platform.

3.

Centrifuge at 18,000 x g for 5 minutes at room temperature. Carefully aspirate and discard
the supernatant using the appropriate glass disposal container. Do not discard down the sink.

4.

Repeat Steps 2 - 3 two more times for a total of 3 treatments.

5.

After the last incubation and centrifugation, carefully remove and discard the supernatant.
Add 1 ml absolute (100%) ethanol to the sample and incubate 10 minutes at room temperature on a rotating platform.

6.

Centrifuge at 18,000 x g for 5 minutes at room temperature. The tissue may be very difficult
to pellet following rehydration. Carefully aspirate and discard the supernatant taking care not
to disturb the sample pellet.

7.

Working at the lab bench, add 1 ml 70% (vol/vol) ethanol solution and incubate 5 minutes atroom temperature on a rotating platform.

8.

Centrifuge at 18,000 x g for 5 minutes at room temperature. Carefully aspirate and discard
the supernatant taking care not to disturb the sample pellet.

9.

Add 1 ml 20% (vol/vol) ethanol solution and incubate 5 minutes at room temperature on arotating platform.

10. Centrifuge at 18,000 x g for 5 minutes at room temperature. Carefully aspirate and discard
the supernatant taking care not to disturb the sample pellet.
11.

Add 1 ml nuclease- and protease-free water and incubate 5 minutes at room temperature on
a rotating platform.

12.

Centrifuge at 18,000 x g for 5 minutes at room temperature. Carefully aspirate and discard
the supernatant taking care not to disturb the sample pellet.

13.

Resuspend the rehydrated tissue in 500 µl FFPE ChIP Buffer containing 5 µl PIC and 5 µl PMSF.

14.

Proceed immediately to Section C: Tissue Homogenization and Chromatin Isolation.
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11Section C. Tissue Homogenization and Chromatin Isolation
The section below describes the solubilization and fragmentation of chromatin using sonication.
Sonication results may vary depending on the quality of the FFPE sample and the sonication
device being used. This protocol has been validated using Active Motif’s EpiShear™ Probe Sonicator in combination with an EpiShear™ Cooled Sonication Platform to maintain probe height andtemperature consistency between samples.
1.

Incubate the rehydrated tissue on a Thermomixer at 50°C and 850 rpm for 1 hour. Then placetubes on ice for at least 2 minutes.

2.

Add 5 µl PIC and 2.5 µl Deacetylase Inhibitor to each tube.

3.

Transfer each sample to a new 2 ml v-bottom microcentrifuge tube.

4.

Sonicate the sample using the EpiShear Cooled Sonication Platform or an ice bath for 30
seconds ON, 30 seconds OFF with a probe tip sonicator at 42% amplitude for 40 minutes
elapsed time (20 minutes total ON time).

5.

Centrifuge at maximum speed for 15 minutes at 4°C. Place a new microcentrifuge tube on ice
during the spin to pre-chill the tube.

6.

Transfer supernatant containing the soluble chromatin into a pre-chilled microcentrifuge
tube. If desired, pool multiple samples from the same tissue together into a single tube
and pipette to mix. Save the pellet in case additional sonication is required to release theinsoluble chromatin from the pellet.
Note:

See Troubleshooting guide in the Appendix for notes to increase solubility.

7.

Label a new 250 µl PCR tube and transfer 20 µl of soluble chromatin for input preparation.
Add 80 µl TE, pH 8.0 to bring the final volume to 100 µl.

8.

Process the Input fractions according to Section D. Store the remainder of each chromatinfraction at -80°C. The Input DNA will be used to quantify the chromatin and evaluate theshearing efficiency.

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12Section D. Input Preparation
1.

To each Input preparation from Step 7 above, add 2 µl RNase A. Cap the PCR tubes andvortex to mix.

2.

Incubate in a thermocycler at 37°C for 30 minutes.

3.

Add 2 µl Proteinase K and 5 µl 5 M NaCl. Cap the PCR tubes and vortex to mix.

4.

Incubate in a thermocycler at 65°C overnight.

5.

Following reversal of cross-links, transfer DNA to a new 1.5 ml microcentrifuge tube. Add 125µl phenol and 64 µl chloroform/isoamyl alcohol (24:1). Shake vigorously for 15 seconds to mix(do not vortex). Incubate at room temperature for 5 minutes.

6.

Centrifuge in a microcentrifuge at maximum speed for 2 minutes. Transfer the aqueous layer(top layer) to a new 1.5 ml microcentrifuge tube. A hazy white layer at interface may still bepresent.

7.

Add 125 µl chloroform/isoamyl alcohol (24:1) to the aqueous layer . Shake vigorously for 15
seconds to mix (do not mix). Incubate at room temperature for 5 minutes.

8.

During the incubation, set up new 1.5 ml microcentrifuge tubes for each ChIP reaction andadd 2 µl Carrier to each tube.

9.

Centrifuge the DNA purifications in a microcentrifuge at maximum speed for 2 minutes.
Transfer the aqueous layer (top layer) to the microcentrifuge tube containing Carrier.

10. Add 300 µl 100% ethanol to the DNA solution and briefly vortex to mix.
11.

Place samples at -80°C for 30 minutes to precipitate the DNA. Alternatively, samples may beleft at -20°C for 2 hours to overnight.

12.

Centrifuge in a microcentrifuge at maximum speed for 15 minutes at 4°C. Mark the tube
where you expect the pellet to form as it may not be visible.

13.

Carefully remove the supernatant taking care not to disturb the location of the DNA pellet.

14.

Add 500 µl 70% ethanol to each tube and invert to mix.

15.

Centrifuge in a microcentrifuge at maximum speed for 5 minutes at 4°C. Mark the tube
where you expect the pellet to form as it may not be visible.

16.

Remove 400 µl supernatant and centrifuge a second time at maximum speed for 2 minutes
at 4°C. Carefully remove as much residual ethanol as possible with a P200 pipet taking care
not to disturb the pellet.

17.

Air-dry the pellet for 10-15 minutes (until residual ethanol has evaporated). Resuspend thepellet in 40 µl FFPE Low-EDTA TE Buffer.

18. Due to the low DNA concentration of the Input material from FFPE chromatin extractions,
we do not recommend analysis of the shearing efficiency by agarose gel electrophoresis.
Instead, we recommend evaluating the quality and quantity of the chromatin by use of afluorometer, such as Qubit. The Nanodrop is not recommended for quantification as the
DNA quantities are usually below the threshold that the Nanodrop can accurately quantify.
www.activemotif.com

13Section E. Analysis of Input DNA and Chromatin Quality
1.

Set up a qPCR reaction to quantify the chromatin Input and to evaluate the solubility of
the chromatin. Use the provided DNA Standards and Human Positive Control GAPDH-2 PCR
Primer Set to analyze the Input DNA. Below is an example qPCR reaction. Please follow
the specific instructions for your qPCR instrument. We recommend using a commerciallyavailable SYBR Green qPCR master mix (e.g. Bio-Rad Cat # 170-8882) and preparing triplicate
reactions.
Reagent

20 µl PCR reactions

2X SYBR Green master mix

10 µl

Primer mix (2.5 µM each primer)*

4 µl

Sterile water

1 µl

DNA sample (DNA Std or Input)

5 µl

Total volume

20 µl* If not using human samples, see notes in Buffer Preparation on page 10.
2.

Place the PCR tubes in a real time PCR instrument. Using the software for your qPCR
machine, assign a value of 2.5 ng/µl to DNA Standard AM1, a value of 0.25 ng/µl to DNA
Standard AM2 and a value of 0.025 ng/µl to DNA Standard AM3. These values will be used toquantify your sample chromatin Input.

3.

Place tubes in a real time PCR instrument and program as below:

		

95°C for 2 minutes(95°C for 15 seconds, 58°C for 20 seconds, 72°C for 20 seconds) for 40 cycles

4.

Include and inspect the melt curve based on the protocols recommended by the qPCR
instrument manufacturer to ensure that primer pairs amplify only a single product.

5.

Your qPCR instrument will assign values (in ng/µl) to each qPCR reaction based on the values
assigned to DNA Standards AM1, AM2 and AM3. If your machine does not average yourtriplicate reactions automatically, you will need to calculate these averages.

6.

For each qPCR reaction you will have used a percentage of your total chromatin. In order tocalculate the total amount of chromatin, multiply the recovered volume of the soluble fraction (~500 µl) by the calculated concentration.

7.

Generate a graph to evaluate the total chromatin yield (ng) for each chromatin preparation(See Figure 1).
a.

A minimum of 200 ng is required for each downstream ChIP reaction, although moreis recommended if possible. Ensure your chromatin preparation contains enoughchromatin to perform ChIP reactions in duplicate with your ChIP antibody of interest.
A positive control H3K9ac antibody is included in the ChIP-IT® FFPE II Kit (Catalog No.
91103) and one positive control ChIP reaction can be performed for each chromatin
preparation.

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14

b.

If enough chromatin is available in the soluble fraction to perform the desired ChIP
reactions, the aliquots stored at -80°C from Section C, Step 8 can be used to performthe ChIP reactions using the ChIP-IT® FFPE II Kit (Catalog No. 53047).

c.

If there is not enough chromatin available in the soluble fraction, additional sonicationof the insoluble pellet from Section C, Step 6 is required. Follow the instructions in Section F: Additional Sonication of Insoluble Pellet.

www.activemotif.com

15Section F. Additional Sonication of Insoluble Pellet
Based on the results of the qPCR analysis in Section E, if there is a significant amount of chromatinremaining in the insoluble pellet, it is important to continue with additional processing of the pellet to release the chromatin. Additional processing of the pellet should also be performed if theyield of the soluble chromatin is too low to proceed with downstream ChIP reactions. A minimumof 200 ng chromatin is required for each ChIP reaction.
For further sonication, the soluble chromatin can be combined with the insoluble pellet and theentire mixture can undergo a second round of sonication and a second analysis by qPCR. This
results in a single soluble and insoluble fraction and is ideal if the sample was overfixed or there
was very little chromatin obtained in the original soluble fraction. One potential issue with thismethod is that further sonication of soluble chromatin may begin to affect the antigenicity of thechromatin, which could affect the quality of the chromatin for use in ChIP.
A second option would be to preserve the original soluble fraction and just process the insoluble
chromatin pellet with a second round of sonication. Following sonication, the two soluble fractions can be combined and quantified by qPCR.
1.

Dilute the insoluble chromatin pellet to a final volume of 500 µl - 1 ml using FFPE ChIP Buffer.
Add 5 µl PIC.

2.

Sonicate the samples using a probe sonicator at 42% amplitude for a 500 µl volume and at
63% amplitude for a 1 ml volume. Sonicate the samples using the EpiShear Cooled Sonication
Platform or an ice bath for 30 seconds on and 30 seconds off for 20 - 40 pulses.

3.

Carefully remove cellular debris by centrifugation at maximum speed for 2 minutes at 4°C.
Collect the supernatant in a new microcentrifuge tube. Resuspend the pellet in 200 µl ChIP
Buffer. Transfer 20 µl of both the soluble (supernatant) and insoluble (pellet) fractions to a
250 µl PCR tube. Label each tube as soluble Input and insoluble Input respectively. Store theremainder of each chromatin fraction at -80°C.

4.

Prepare the Input DNA according to Section D. Then proceed to Section E for analysis of thesoluble and insoluble fractions by qPCR.

www.activemotif.com

16

Appendix
Section G: qPCR Primer Design
A. Design of the primers• Design and analyze your potential primer pairs using an in silico PCR program (i.e. Primer3
at http://frodo.wi.mit.edu/ or the UCSC Genome Browser at http://genome.cse.ucsc.edu/
cgi-bin/hgPcr).
• Primers that dimerize should be avoided, as they will be bound by SYBR Green, which willcompromise accurate quantitation. You can test your primers for self-complementarity andsecondary structure at http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi.
• Ideally, the amplicons should be 75-150 bp in length.
• For use with the ChIP-IT qPCR Analysis Kit, primers should be designed to anneal optimally at
58°C with a recommended length of 18-22 bp.
• Active Motif offers ChIP Control qPCR primer sets validated to work in our ChIP-IT qPCR
Analysis Kit. To see a list of the available species-specific primers, please visit
www.activemotif.com/chipprimers.

www.activemotif.com

17Section H. Troubleshooting Guide
Problem/question

Recommendation

At what points in the
protocol can I stop?

The protocol may be stopped and samples stored at the times and temperatures below:
1. After sonication (C.6), -80°C.
4. During Input preparation DNA purification (D.11), -80°C or -20°C.

My sample does not pellet
during tissue rehydration

Tissue may be difficult to pellet following rehydration. Carefully remove supernatant anddiscard. Continue to repeat centrifugation steps until the tissue is primarily left as a pellet
with little to no liquid.

Difficulty with homogenization of tissue

Samples may behave differently during homogenization depending on the fixation conditions used to prepare the FFPE samples, the type of tissue you are working with or due
to the differences between normal and tumor tissues. It may be necessary to combine
multiple tubes of the same sample together and use a tissue tearor for 40 seconds tohelp disrupt the cells.
It is recommended to perform dounce homogenization following use of a tissue homogenizer. Use a dounce homogenizer with a small clearance pestle (Active Motif Catalog
Nos. 40401 & 40415). If the material is difficult to work with, start with a loose pestle tobreak up the cells and then switch to the tight-fitting pestle.

Low solubility of chromatin following sonication

Sonication samples were emulsified. Avoid emulsification by turning up the power ofthe sonicator gradually. If a chromatin preparation becomes emulsified inadvertently,
discontinue shearing and centrifuge the sample for 4 minutes at 8,000 rpm in a 4°C
microcentrifuge to remove trapped air.

Low chromatin yield

Poor quality FFPE samples. Depending on the formalin fixation and storage conditions,
FFPE samples may experience degradation and loss of antigenicity over time. Thesesamples may not be suitable for chromatin extraction and downstream ChIP analysis.
Consider using alternative sample material.
Consider using additional tissue material and repeating the chromatin preparation.
Depending on the size and quality of the sample it may be difficult to obtain sufficientchromatin from 3 - 5 slides/sections.
Additional processing of the insoluble pellet may be necessary to obtain additional chromatin. Follow the recommendations in Section F to dilute the insoluble pellet. Repeat thesonication with an additional 20-40 pulses. Evaluate the secondary sonication by qPCR.

www.activemotif.com

18Section I. Related Products
ChIP-IT® Kits		

FormatCatalog No.

ChIP-IT® Express		ChIP-IT® Express Enzymatic		
ChIP-IT® Express Shearing Kit		
ChIP-IT® Express Enzymatic Shearing Kit		
ChIP-IT® High Sensitivity		
Low Cell ChIP-Seq Kit		
ChIP-IT® qPCR Analysis Kit		
ChIP-IT® ChIP-Seq		
ChIP-IT® FFPE II		
ChIP-IT® FFPE Chromatin Preparation Kit		
ChIP-IT® Express HT		Next Gen DNA Library Kit		
Re-ChIP-IT®		RNA ChIP-IT®		
Chromatin IP DNA Purification Kit		
EpiShear™ Probe Sonicator		
EpiShear™ Cooled Sonication Platform, 1.5 ml		
ChIP-IT® Protein G Magnetic Beads		
Protein G Agarose Columns		
Siliconized Tubes, 1.7 ml		
ChIP-IT® Control qPCR Kit – Human		
ChIP-IT® Control qPCR Kit – Mouse		ChIP-IT® Control qPCR Kit – Rat		ChIP-IT® Control Kit – Human		
ChIP-IT® Control Kit – Mouse		ChIP-IT® Control Kit – Rat		
Ready-to-ChIP HeLa Chromatin		
Ready-to-ChIP Hep G2 Chromatin		
Ready-to-ChIP K-562 Chromatin		
Ready-to-ChIP NIH/3T3 Chromatin		Bridging Antibody for Mouse IgG		
Dounce Homogenizer		Dounce Homogenizer		

25 rxns
25 rxns
10 rxns
10 rxns
16 rxns
16 rxns
10 rxns
10 libraries
16 rxns
5 rxns96 rxns
16 rxns
25 rxns
25 rxns
50 rxns
110 V
1 platform
25 rxns
30 rxns
25 tubes
5 rxns
5 rxns
5 rxns
5 rxns
5 rxns
5 rxns
10 rxns
10 rxns
10 rxns
10 rxns
500 µg
1 ml
15 ml

53008
53009
53032
53035
53040
53084
53029
53041
53047
53030
53018
53216
53016
53024
58002
53051
53080
53014
53039
53036
53026
53027
53028
53010
53011
53012
53015
53019
53020
53021
53017
40401
40415

ChIP-validated Antibodies
For an up-to-date list of over 125 ChIP-validated antibodies, please visit www.activemotif.com/chipabs.
Whole Genome Amplification		

FormatCatalog No.

GenoMatrix™ Whole Genome Amplification Kit		

1 kit

58001

Co-Immunoprecipitation		

FormatCatalog No.

Nuclear Complex Co-IP Kit		
Universal Magnetic Co-IP Kit		
Modified Histones Array		50 rxns
25 rxns
Format

54001
54002
Catalog No.

MODified™ Histone Peptide Array		

1 array

13001

Histone Modification FP Binding Assay		

FormatHiLite™ Histone H3 Methyl-Lys9 / Lys27 FP Binding Assay

1 kit

www.activemotif.com

19

Catalog No.
57001 Technical Services

Technical Services
If you need assistance at any time, please call Active Motif Technical Service at one of thenumbers listed below.
Active Motif North America
1914 Palomar Oaks Way, Suite 150
Carlsbad, CA 92008
USA
Toll Free:877 222 9543
Telephone: 760 431 1263
Fax:
760 431 1351
E-mail:tech_service@activemotif.com
Active Motif Europe
Avenue Reine Astrid, 92
B-1310 La Hulpe, Belgium
UK Free Phone:		
0800 169 31 47
France Free Phone:
0800 90 99 79
Germany Free Phone: 0800 181 99 10
Telephone: 		+32 (0)2 653 0001
Fax: 		
+32 (0)2 653 0050
E-mail: 		
eurotech@activemotif.com
Active Motif Japan
Azuma Bldg, 7th Floor
2-21 Ageba-Cho, Shinjuku-Ku
Tokyo, 162-0824, Japan
Telephone: +81 3 5225 3638
Fax:
+81 3 5261 8733
E-mail:japantech@activemotif.com
Visit Active Motif on the worldwide web at http://www.activemotif.com
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