TAM-ChIP Assay Reagents(version A2)

Catalog No. 53128

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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
Flowchart of Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Kit Performance and Benefits  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Protocol Overview and Time Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Kit Components and Storage
TAM-ChIP Assay Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Additional Materials Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Protocols – Experimental Set Up
Cell Growth and Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Buffer Preparation  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  10
Protocols – Preparation of Sheared Chromatin
Section A. Cell Fixation Starting with Cultured Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Section B. Chromatin Sonication of Cultured Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Section C. Cell Fixation Starting with Fresh or Frozen Tissue . . . . . . . . . . . . . . . . . . . . . . . . 15
Section D. Chromatin Sonication of Tissue  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  16
Protocols – Chromatin Immunoprecipitation
Section E. Immunoprecipitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  19
Protocols – ChIP DNA Purification
Section F. Reversal of Cross-links and DNA Purification . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Protocols – Library Amplification & Size Selection
Section G. Library Amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Section H. Size Selection  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
Section I. Sequencing Guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
Appendix
Section J. Troubleshooting Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
Section K. Related Products  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Technical Services  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

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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 patterns of proteinsbound to specific DNA loci. When used in combination with whole-genome sequencing, insights
into gene regulation, gene expression, mechanisms of chromatin modification and pathway analysis are possible.
Traditional methods to prepare ChIP-Seq libraries involve a series of processing steps to end repair
the DNA into phosphorylated blunt ends, followed by addition of an ‘A’ base to the 3´end of the
DNA fragments for ligation to adapters containing single ‘T’ base overhangs. Libraries are then PCR
amplified and size-selected prior to Next-generation sequencing (NGS). In addition to being time
consuming, one of the biggest challenges associated with traditional library preparation is the
sample loss that can occur throughout the multi-step process. To overcome this obstacle, Active
Motif has developed TAM-ChIP*, for simultaneous genomic targeting and library preparation.
Active Motif’s TAM-ChIP uses antibody/oligonucleotide conjugates to target a unique TsTn5transposase to genomic regions containing a particular mark of interest (histone or transcription
factor). Upon antibody binding, the transposase cuts the nearby DNA and pastes the antibody
associated oligonucleotides into the DNA sequence. These oligonucleotides contain the necessary adapters for library amplification and NGS using Illumina® platforms, thereby eliminating
multiple end-repair and ligation steps associated with traditional library preparation. Additionally,molecular identifiers (8 bp random N sequence) are included in the adapter sequences to easily
remove PCR duplicates to avoid PCR bias. Another benefit of TAM-ChIP is that the combination ofchromatin fragmentation and transposase tagmentation creates shorter DNA fragments for higher
resolution of protein-DNA binding sites.
TAM-ChIP Assay Reagents includes the necessary buffers and PCR amplification reagents needed
to perform 16 TAM-ChIP reactions. Additional reagents that are required, but not included are a
ChIP-Seq validated antibody for the target protein of interest and the appropriate species-specific
TAM-ChIP antibody conjugate.

product

formatcatalog no.

TAM-ChIP Assay Reagents

16 rxns

53128

TAM-ChIP anti-rabbit conjugate

16 rxns

53126

TAM-ChIP anti-mouse conjugate

16 rxns

53127* Patent pending

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Overview of TAM-ChIP

Overview of the TAM-ChIP process.
TAM-ChIP is designed to perform antibody-based genomic targeting and NGS library preparation at once. First, intact cells
are fixed with formaldehyde, which cross-links and preserves protein/DNA interactions. DNA is then sheared into small
fragments using sonication and incubated with a ChIP-validated antibody directed against the DNA-binding protein ofinterest. Then, a species-specific TAM-ChIP antibody conjugate containing Illumina-compatible sequencing adapters andrandom molecular identifier is added to bind the ChIP antibody. The antibody-bound protein/DNA complexes are immunoprecipitated with Protein G agarose beads and washed to remove non-specific DNA. Activation of the TsTn5 transposase
cuts the nearby DNA surrounding the genomic region of interest and pastes the antibody associated barcoded adapters
into the DNA sequences. Following immunoprecipitation, cross-links are reversed, the proteins are removed by Proteinase
K, and the DNA is recovered and purified. ChIP enriched DNA is now ready for library amplification and sequencing.

TAM-ChIP Flowchart (opposite page; also available on www.activemotif.com/tamchip and click the Methods tab)
TAM-ChIP is a multi-step process that involves assembling the unique TsTn5 transposase to the secondary antibody
conjugate and an oligonucleotide prior to immunoprecipitation (A). The TAM-ChIP anti-species antibody conjugatecontains a single-stranded oligonucleotide comprised of the i5 antibody index for Next-generation sequencing. Eachanti-species antibody has its own i5 index to enable multiplexing of both the anti-rabbit and anti-mouse conjugates within
the same sequencing reaction. When the antibody conjugate is combined with a single-stranded pMENTS oligonucleotide,the complementary sequences anneal to create a double-stranded recognition sequence for the TsTn5 transposase toassemble. This process loads inactivated transposase onto the antibody conjugate. At the same time, a single-stranded
oligonucleotide is also annealed to pMENTS and loaded with inactive TsTn5.
During immunoprecipitation (B), the ChIP-validated antibody of interest is combined with the sonicated chromatin andincubated overnight. The next day, the assembled TAM-ChIP conjugate is added to the immunoprecipitation reaction.
Following an incubation, protein G agarose beads are added to capture the chromatin fragments of interest. Each ChIP reaction is loaded onto a ChIP filtration column and the oligonucleotide assembled with transposase is added to the column.
The TsTn5 is activated to insert the i5 index and oligonucleotide sequence into the genomic region surrounding the protein
of interest. Following elution and DNA purification, the ChIP library is ready for amplification.
The TAM-ChIP library is amplified by PCR prior to sequencing (C). During PCR, the second strand of the DNA template isextended. Then, the i7 TAM-ChIP Index containing the 3 bp sample barcode and 8 bp random molecular identifier (MID)for PCR de-duplication is incorporated into the template. Additional PCR cycles amplify the entire template using theamplification primer mix. Libraries are size-selected and sequenced using Illumina platforms.

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Flowchart
TAM-ChIP FlowchartA

TAM-ChIP Oligo Assembly

TAM-ChIP Antibody Conjugate Assembly

ssDNA Oligonucleotide

ssDNA Antibody index (i5)pMENTS

pMENTS
Incubate 30 min at RT

Incubate 30 min at RT

TsTn5 transposase

TsTn5 transposase

Incubate 30 min at RT

Incubate 30 min at RT

ssDNA Antibody index (i5)pMENTS
TsTn5 transposase

ssDNA Oligonucleotide
pMENTS
TsTn5 transposase

TAM-ChIP ImmunoprecipitationB

A ChIP-validated antibody of interest is incubatedwith sonicated chromatin overnight.

ssDNA Antibody index (i5)pMENTS
TsTn5 transposase

The TAM-ChIP Antibody conjugate is added
to the immunoprecipitation reaction.

ssDNA Antibody index (i5)Protein G beads

pMENTS
TsTn5 transposase

ssDNA Oligonucleotide
pMENTS
TsTn5 transposase

Protein G agarose beads are added to
the reaction and the entire complex isloaded into a ChIP filtration column.
Next, the oligonucleotide assemblyis added to the filtration column andthe TsTn5 transposase is activated.

C

TAM-ChIP Library Amplification
Antibody index (i5)DNA of interest

pMENTS

pMENTS

Oligonucleotide

During PCR cycle 1, elongation fills in the complementary sequence

Antibody index (i5)DNA of interest

pMENTS

pMENTS

Oligonucleotide

During PCR cycle 2, elongation continues with the TAM-Index (i7)Antibody index (i5)DNA of interest

pMENTSTAM-Index (i7) with random
MID and sample barcode

PCR cycles 3-14 amplify the DNA using the amplification primer set

Amplification primer 1
Antibody index (i5)DNA of interest

pMENTSTAM-Index (i7)Amplification primer 2*Only a single strand is depicted for convenience. Analogous chemistry is carried out on the complementary strand.

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3Kit Performance and Benefits
TAM-ChIP Advantages:• Tagmentation of the DNA using the TsTn5 transposase aids in chromatin fragmentation andprovides higher resolution of protein binding sites• Avoid sample loss common with traditional library preparation steps• Molecular identifiers (8 bp random barcodes) enable distinction of PCR duplicates from
biological replicates during analysis to increase the number of unique alignments 3-fold• Highly robust procedure has been validated across multiple sample types using both transcription factor and histone marks

Figure 1: Sequencing data comparing TAM-ChIP replicates to traditional ChIP-Seq peaks.
TAM-ChIP was performed using 10 µg chromatin from MCF-7 cells with antibodies to CTCF and Histone H3K4me3. Results
show reproducibility between replicates and peaks that are consistent with published ENCODE data sets.

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4ChIP-Seq

TAM-ChIP

Fragment size determined by sonication

Fragment size determined bysonication + TsTn5 tagmentation

Fragment length

Peak width

ChIP-Seq

TAM-ChIP

Fragment Length (nt)

250

125

Peak Width (nt)

1245

170

Figure 2: TAM-ChIP provides greater binding site resolution.
TAM-ChIP utilizes a combination of sonication and TsTn5 tagmentation to cut the DNA surrounding the genomic regionof interest. The addition of tagmentation produces smaller DNA fragments than traditional ChIP-Seq, which improves theresolution of protein-DNA binding sites.

Protocol Overview and Time Table
Required Time
Cell or Tissue Fixation and Lysis

1.5 hours

Chromatin Sonication

15 minutes per sample

Assessment of Chromatin Size*

4.5 hours for cell culture
Overnight for tissue

Immunoprecipitation

Overnight

Assembly of Antibody Conjugate

1.5 hours

Binding of TAM-ChIP Conjugate

2 hours

Binding to Protein G agarose Beads

2 hours

Wash Immune Complexes

30 minutes

Activate TsTn5 transposase

1 hourReversal of Cross-links

2 hours

DNA Purification

30 minutes

Library Amplification & Size Selection

2 hours* The protocol varies between cell culture and tissue samples.
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5Kit Components and Storage
Please store each component at the temperature indicated in the table below.
Reagents

Quantity

Storage

RNase A (10 µg/µl)

40 µl

-20°C

Proteinase K (10 µg/µl)

200 µl-20°C

Blocker

100 µl-20°C

100 mM PMSF

500 µl-20°C

Protease Inhibitor Cocktail (PIC)

500 µl-20°C

Precipitation Buffer

1.5 ml-20°C

Carrier

35 µl

-20°C

10X PBS

120 ml-20°C

1 M DTT

80 µl

-20°C

10X Elution Buffer AM2

250 µl-20°C

Q5 High-Fidelity Polymerase (2 U/µl)

10 µl

-20°C

5X Q5 Reaction Buffer

2 x 130 µl-20°C

dNTPs (10 mM)

40 µl

-20°C

Fixation Buffer

2 x 1.5 ml

4°C

Protein G Agarose Beads*

500 µl4°C

TE pH 8.0

3 x 1.5 ml

RT

5 M NaCl

400 µlRT

Detergent

25 ml

RT

Stop Solution

20 ml

RT

Chromatin Prep Buffer

85 ml

RT

ChIP Filtration Columns

16 ea

RT

Caps and plugs for columns

50 ea

RT

ChIP Buffer

35 ml

RT

ChIP Buffer without SDS

10 ml

RT

Wash Buffer AM1

100 mlRT

Elution Buffer AM4

2 x 1.5 ml

RT

5 ml

RT

DNA Purification Elution Buffer* The Protein G Agarose Beads are shipped on dry ice and can be stored frozen until their first use.
Once thawed, the Protein G beads should not be re-frozen, but stored at 4ºC.
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Additional materials required but not included• A ChIP-Seq validated antibody directed against the protein of interest• TAM-ChIP antibody conjugate appropriate for the ChIP-seq validated antibody species used
for immunoprecipitation (Catalog Nos. 53126 and 53127)• Dounce homogenizer with a small clearance pestle (e.g. Active Motif Catalog Nos. 40401 &
40415) with the tight-fitting “A” pestle). Use of a homogenizer is necessary to prepare cells
for chromatin shearing.
• 37% formaldehyde solution with 10-15% methyl alcohol to prevent polymerization (e.g. Sigma
Aldrich Catalog No. 252549). Do not use paraformaldehyde.
• Phenol, saturated (DNA Purification, Molecular Biology Grade, Amresco Catalog No. 0945)• Chloroform/isoamyl alcohol (24:1) (DNA Purification, Molecular Biology Grade)• Phase Lock Gel™ Heavy tubes (VWR, Catalog No. 10847-802)• 100% ethanol (absolute)• 80% ethanol• Agencourt AMPure XP Beads (Beckman Coulter, A63880)• DNase-free H2O• Rocking platform for culture plates• 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)• Microcentrifuge (table top centrifuge 4°C) and microcentrifuge tubes• 250 µl PCR tubes• Thermocycler• 15 and 50 ml conical tubes• DNA quantitation apparatus• 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))• Agarose gel electrophoresis apparatus or method for visualizing fragmentation• Razor blades (for tissue preparations)• Hand-held homogenizer for tissue preparations (e.g. Biospec Products Tissue-Tearor)• Cell scraper (rubber policeman)www.activemotif.com

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Protocols – Experimental Set Up
PLEASE READ THE ENTIRE PROTOCOL BEFORE STARTING!
Cell Growth Recommendations
When planning an experiment, calculate the number of chromatin preparations you will requireand determine the number of ChIP reactions you plan to perform on each chromatin preparation.
Be sure to include the appropriate positive and negative control ChIP reactions in your calculations. Also, note that if you wish to analyze the effect of particular compounds or culturingconditions on transcription factor/DNA interactions, you should prepare chromatin from control(untreated) cells as a reference sample. The minimum recommended number of cells that should
be used for the preparation of chromatin is 100,000 cells.
6-well
plate

60 mm
dish

100 mmdish

150 mmdish

Seeding Density

0.3 x 106

0.8 x 106

2.2 x 106

5.0 x 106

Cells at 70-80%
Confluency*

0.9 x 106

2.4 x 106

6.6 x 106

15.0 x 106

Growth Medium
Volume

3 ml

5 ml

10 ml

20 ml

Cell Fixative Solution

300 µl500 µl

1 ml

2 ml

Stop Solution

165 µl

275 µl

550 µl1.1 mlPBS Wash Buffer(used per wash)

1 ml

2 ml

5 ml

10 ml

Chromatin Prep Buffer

1 ml

2 ml

5 ml

5 ml

ChIP Buffer

500 µl500 µl500 µl500 µl* The number of cells on a confluent plate or dish will vary with cell type. For this table, HeLa
cells were used. Please adjust as needed based on your particular cell type.
**Please refer to the descriptions below for complete details on buffer preparations

Buffer Preparation
Complete Cell Fixation Solution
Buffer should be prepared fresh before each experiment. For every 20 ml of cell growth medium
used, prepare 2.5 ml of Complete Cell Fixation Solution by adding 180 µl Fixation Buffer to 1.57 ml
sterile water in a 15 ml conical tube. Using appropriate precautions (i.e. safety glasses, gloves andlab coat), add 750 µl 37% formaldehyde to the tube and vortex to mix. Use 1/10 growth medium
volume per plate. Complete cell fixation solution can be added to the growth medium in the presence or absence of serum.
Complete Tissue Fixation Solution
Buffer should be prepared fresh before each experiment. Prepare 10 ml of Tissue Fixation
Solution for each tissue sample to be processed by adding 1 ml 10X PBS to 8.7 ml sterile water in a
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15 ml conical tube. Using appropriate precautions (i.e. safety glasses, gloves and lab coat), add
280 µl 37% formaldehyde to the tube and vortex to mix.
Stop Solution
Is provided ready to use. Use 1/20 media volume per cell culture plate or 515 µl per 10 ml Complete
Tissue Fixation Solution.
PBS Wash Buffer
Prepare 25 ml PBS Wash Buffer for every 15 cm plate or tissue sample. To a 50 ml conical tube add
21.25 ml sterile water, 2.5 ml 10X PBS and 1.25 ml Detergent. Mix by inverting. Place PBS Wash Buffer
on ice to chill. PBS Wash Buffer can be prepared in large quantities and stored at 4ºC for 6 months.
100 mM PMSF and Protease Inhibitor Cocktail (PIC)
Thaw the PMSF and 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.
1 M DTT
Prepare a fresh 70 mM DTT working stock for each TAM-ChIP experiment. Take 5 µl 1M DTT and
add to 66 µl sterile water. Vortex to mix. This makes a 70 mM working solution. Store any unused
solution at -20ºC. Discard at the end of the day.
Chromatin Prep Buffer
Is supplied ready to use.
ChIP Buffer without SDS
Is supplied ready to use.
ChIP Buffer
Is supplied ready to use.
Protein G Agarose Beads
The supplied agarose beads require washing prior to use. Follow the instructions in the manual towash the beads for use in the ChIP reactions. There is no need to pre-block the beads or pre-clear
the sample. For best results, gently shake and invert the tube to resuspend the agarose beads. The
beads settle quickly, and therefore should be resuspended just before pipetting. We recommend
cutting 2 mm from the end of a pipet tip prior to pipetting to prevent the tip from becoming
clogged. Protein G Agarose Beads are shipped on dry ice and can be stored frozen until theirfirst use. Once thawed, beads should not be re-frozen by the customer. Protein G Agarose
Beads should be stored at 4ºC.

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Recommendations
ChIP-validated Antibody
We recommend using 4 µg antibody per ChIP reaction. However, this will vary according to the affinity of the antibody and the quality of the chromatin; you may need to use more of a particularantibody. ChIP antibodies must recognize fixed, native protein that is bound to DNA and/or
complexed with other proteins. Many antibodies that perform well in other applications do not
perform in ChIP. Thus, ChIP performed with an antibody that has not been ChIP-validated must
include appropriate controls. To see a list of available ChIP-validated antibodies available from
Active Motif, please visit www.activemotif.com/chipabs.
Chromatin Shearing Tips
We suggest using a probe sonicator (i.e. Active Motif’s EpiShear Probe Sonicator) which employs adirect sonication method to prepare chromatin for use in TAM-ChIP. Indirect sonication systems may
require longer sonication times to achieve optimal chromatin shearing. ChIP experiments usuallyrequire chromatin that has been sheared to a size of 200-1000 bp. In general, shearing efficiency isimproved 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 withair bubbles. To determine the appropriate shearing level for your sample, 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 the highest 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 of chromatin, samples should be kept
on ice as much as possible during shearing, and shearing should be performed discontinuously (i.e.
sonicate for 20 seconds, then place on ice/water for 30 seconds, sonicate again for 20 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
It is recommended to use 5-10 µg chromatin per IP reaction (0.75-1.5 million cell equivalents).
Safety Precautions
Formaldehyde and PMSF are highly toxic chemicals. Appropriate safety precautions (i.e. safety
glasses, gloves and lab coat) should be used. Also, formaldehyde is highly toxic by inhalation andshould be used only in a ventilated hood. Finally, chromatin sonication should be performed in abiosafety hood if the chromatin is extracted from biohazardous or infectious materials.

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Protocols – Preparation of Sheared Chromatin
Section A: Cell Fixation Starting with Cultured Cells
This protocol describes cell fixation and chromatin preparation from one 15 cm plate (approximately 1.5 x 107 cells). We recommend using 20 ml growth medium per 15 cm plate. Please refer topage 7 for information on scaling the protocol for use with other amounts of cells. The minimum
cell number to be used for the preparation of chromatin is 100,000 cells.
1.

Prepare 15 cm plates for each cell line to be tested. Grow the cells to 70-80% confluency.
Stimulate cells as desired to activate the pathway of interest.

2.

Freshly prepare Complete Cell Fixation Solution for each 15 cm plate. The volumes listed
below are enough to process one 15 cm plate. Please refer to the chart on page 8 to scale thesolution volumes.

3.

To fix cells, add 1/10 growth medium volume of freshly prepared Complete Cell Fixative Solution to the existing culture media for the cells (e.g. 20 ml growth medium would get
2 ml Complete Cell Fixation Solution). Shake gently at room temperature for 15 minutes.

4.

Stop the fixation reaction by adding 1/20 media volume of Stop Solution to the existing
culture media for the cells (e.g. 20 ml growth medium would get 1.1 ml Stop Solution). Swirlto mix and incubate at room temperature for 5 minutes.

5.

Following the incubation, hold the plate at an angle and using a rubber policeman scrape
cells down to collect them at the bottom edge of the plate. Use a pipette to transfer thecells to a 50 ml conical tube on ice.

6.

Pellet the cells from step 5 by centrifugation for 3 minutes at 1,250 x g at 4°C.

7.

Remove the supernatant and discard. Resuspend the pellet(s) in 10 ml ice-cold PBS Wash Buffer by pipetting up and down. Keep samples ice-cold for the remainder of the procedure.

8.

Centrifuge for 3 minutes at 1,250 x g at 4°C. Remove the supernatant and discard. Wash thepellet(s) a second time in 10 ml ice-cold PBS Wash Buffer by pipetting up and down. Centrifuge for 3 minutes at 1,250 x g at 4°C. Remove the supernatant and discard. Cell pellets may
be stored at -80°C at this stage.

9.

Resuspend each pellet(s) in 5 ml Chromatin Prep Buffer supplemented with 5 µl PIC and5 µl 100 mM PMSF. Pipet up and down to mix.

10. Incubate on ice for 10 minutes.
11.

Transfer the resuspended pellets individually to a chilled dounce homogenizer on ice. Use
the tight fitting pestle (Type A) to homogenize the sample for 30 strokes. Transfer the contents to a new 15 ml conical tube and centrifuge for 3 minutes at 1,250 x g at 4°C.
Monitor Cell Lysis: To ensure cell lysis, take 10 µl of the cell lysate from the dounce and lookat it under a phase contrast microscope using a hemocytometer to verify that the nuclei
have been released. It is often helpful to look at the cells before and after the lysis step as
this makes it easier to identify the nuclei versus whole cells. Intact cells should have a dark
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nuclei will appear as dots surrounded by asymmetric debris. If the cells are not lysed, thendounce on ice with an additional 10 strokes, or until the cells are lysed.
12.

Remove the supernatant and discard. Resuspend each pellet in 500 µl ChIP Buffer supplemented with 5 µl PIC and 5 µl 100 mM PMSF. Transfer the contents to a new 2 ml microcentrifuge tube.

13.

Incubate on ice for 10 minutes. Proceed to Step B: Chromatin Sonication of Cultured Cells.

Section B. Chromatin Sonication of Cultured Cells
The section below describes the fragmentation of chromatin using sonication. Sonication results
may vary depending on cell type and sonication device being used. This protocol has beenvalidated using Active Motif’s EpiShear™ Probe Sonicator in combination with an EpiShear™ Cooled
Sonication Platform to maintain probe height and temperature consistency between samples. We
do not recommend sonication of samples containing less than 100,000 cells and/or 350 µl volume.
The ChIP Buffer has been optimized for immunoprecipitation performance, however, due to its
unique composition optimization of sonication conditions may be required. To maintain the highsensitivity of the assay, we recommend using our buffer system and altering the sonication time
and/or amplitude of your sonication system to achieve the desired fragmentation (e.g. somesystems may require as much as a three-fold increase in sonication time to improve chromatin
shearing). Please pay particular attention to our protocol regarding the processing of input chromatin for agarose gel analysis prior to the chromatin immunoprecipitation reaction as many steps
may differ from traditional ChIP protocols and failure to follow the outlined procedure may leadto artifacts in the gel images as shown in Figure 3 on page 14.
1.

Place the 2 ml microcentrifuge tube containing the chromatin into the tube cooler or packed
ice. Open cap and submerge the microtip into the liquid until the microtip is approximately
5 mm from the bottom of the tube. Sonicate according to optimized settings for the cell
type being used (see Recommendations on page 10). A recommended starting range forcultured cells is: 25% amplitude, pulse for 30 seconds on and 30 seconds off for a total
sonication “on” time of 10 minutes (or 20 minutes elapsed time).

2.

Spin tubes at 4°C in a microcentrifuge at maximum speed for 2 minutes to pellet the cellulardebris.

3.

Transfer 25 µl of each chromatin preparation into a 250 µl PCR tube for analysis of shearing
efficiency and chromatin quantification.

4.

Aliquot the remainder of each chromatin preparation into 1.5 ml microcentrifuge tubes. We
recommend making aliquots of 150 µl volume and storing at -80°C.
Note:

The size of the chromatin sonication should be verified before proceeding to theimmunoprecipitation step.

www.activemotif.com

12Chromatin Quantification
5. To each 25 µl chromatin preparation from Step 3 above, add 175 µl TE pH 8.0 and
1 µl RNAse A. Cap the PCR tubes and vortex to mix
6.

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

7.

Add 2 µl Proteinase K to each tube and vortex. Incubate tubes in a thermocycler at 55°C for
30 minutes and then increase the temperature to 80°C for 2 hours.

8.

Transfer each chromatin input to a 1.5 ml microcentrifuge tube. Add 83 µl Precipitation Buffer, 2 µl Carrier and 750 µl absolute ethanol. Vortex to mix and chill at -80°C for 30 minutes
to overnight.

9.

Spin tubes at 4°C in a microcentrifuge at maximum speed for 15 minutes.

10. Carefully remove the supernatant taking care not to disturb the pellet. Wash the pellet with
500 µl 70% ethanol and spin at 4°C in a microcentrifuge at maximum speed for 5 minutes.
11.

Carefully remove the supernatant taking care not to disturb the pellet. Remove residual
ethanol with a pipet tip. Leave the tubes uncapped and air dry for 10-15 minutes.

12.

When the pellets are dry, add 25 µl DNA Purification Elution Buffer to each tube. Incubate atroom temperature for 10 minutes. Then vortex to ensure the pellet is completely resuspended. This solution contains your DNA for quantification.

13.

Read the absorbance of each sample on a NanoDrop or other spectrophotometer at
260 nm to determine the DNA concentration of each chromatin preparation. Set aside
500 ng of DNA for analysis as described in Step 14. Store the remaining DNA at -20°C.

14.

Analyze each chromatin preparation on an agarose gel by following the instructions below.
a.

Prepare 500 mM NaCl by adding 2 µl 5M NaCl to 18 µl sterile water. Vortex to mix.

b.

Transfer 500 ng of Input DNA to a 250 µl PCR tube and add 1 µl 500 mM NaCl. Adjust
the final volume to 10 µl with sterile water if needed.

c.

Heat samples in a thermocycler at 100°C for 20 minutes followed by ramping the temperature down to 50°C.

d.

Remove tubes from the thermocycler and incubate at room temperature for 5 minutes.

e.

Add gel loading buffer to each sample and run on a 1.5% agarose gel. Include 100 bp
and 1 kb DNA ladders to analyze chromatin size. DNA should appear as a smear anywhere between 200-1000 bp.

Note:

Chromatin may look different on an agarose gel compared to chromatin prepared using traditional ChIP methods. However, this will not affect the sensitivityof the assay or increase background signal. Please follow the protocol as listed
above for preparing Input DNA. Use of an alternative reverse cross-linking
method or omitting the 20 minute incubation at 100°C in NaCl is not recommended as this will cause artifacts that make the DNA appear larger. As long as
the chromatin falls within the recommended 200-1000 bp range, proceed with
the ChIP reaction. If fragments do not fall within this range sonication conditions
should be further optimized.

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13

15.

If chromatin preparations were successful, the aliquots stored at -80°C from Section B, Step 4
can be used to perform the ChIP reactions in Section E.

Figure 3: Validation of chromatin shearing efficiency following reversal of cross-links at 80°C for 2 hours.
Chromatin preparations of MCF-7 cells were fixed and sonicated using the EpiShear™ Probe Sonicator and EpiShear™
Cooled Sonication Platform from Active Motif. Input DNA was prepared in duplicate according to Section B, Steps 5-13 in
the manual. In Sample 1, Step 14 was not performed and 500 ng of input DNA was loaded directly onto a 1.5% agarose gel
without receiving the addition of NaCl and incubation at 100°C. The omission of Step 14 has caused a buffer artifact thatmakes the DNA appear larger on a gel. The duplicate sample, Sample 2, was processed according to the manual instructions
and included the addition of NaCl and incubation at 100°C as stated in Step 14. Analysis of 500 ng of this input DNA on a
1.5% agarose gel shows the expected fragmentation between 200-1000 bp. The difference in DNA sizing on the gel between
the two samples illustrates the importance of following the protocol recommendations regarding the processing of input
chromatin for agarose gel analysis prior to chromatin immunoprecipitation. Omission of key steps can lead to inaccurate
analysis of chromatin shearing efficiency. If the protocol steps were followed and the DNA fragments fall outside of therecommended range, sonication conditions should be further optimized.

www.activemotif.com

14Section C: Cell Fixation Starting with Fresh or Frozen Tissue
This protocol describes cell fixation and chromatin preparation from 100-400 mg fresh or frozen
animal tissue. If performing chromatin preparation on multiple tissue samples, we recommend
completing Steps 1-7 for each sample before processing the next sample.
1.

For tissue fixation, transfer 10 ml Complete Tissue Fixation Solution (see Buffer Preparation
on page 8) to a 60 mm petri dish. Place the dish on ice.

2.

Add 100-400 mg fresh or frozen tissue sample to the petri dish and ensure that the sample isfully immersed. Cut the tissue sample into small pieces (approximately 1 mm cubes) using arazor blade.

3.

Transfer the sample plus the Complete Tissue Fixation Solution to a 15 ml conical tube androtate at room temperature for 15 minutes.

4.

Stop the fixation reaction by adding 515 µl Stop Solution to the conical tube and rotate atroom temperature for 5 minutes.

5.

Place the conical tube on ice and homogenize the contents with a hand-held tissue homogenizer set at 30,000 rpm for 45 seconds.

6.

Pellet the cells from Step 5 by centrifugation for 3 minutes at 1,250 x g at 4°C.

7.

Remove the supernatant and discard. Resuspend the pellet in 10 ml ice-cold PBS Wash Buffer
by pipetting up and down. Keep samples ice-cold for the remainder of the procedure.

8.

Centrifuge for 3 minutes at 1,250 x g at 4°C. Remove the supernatant and discard. Wash thepellet(s) a second time in 10 ml ice-cold PBS Wash Buffer by pipetting up and down. Centrifuge for 3 minutes at 1,250 x g at 4°C. Remove the supernatant and discard. (Cell pellets may
be stored at -80°C at this stage).

9.

Resuspend each pellet in 5 ml Chromatin Prep Buffer supplemented with 5 µl PIC and5 µl 100 mM PMSF.

10. Incubate on ice for 10 minutes.
11.

Transfer the resuspended pellet(s) individually to a chilled dounce homogenizer on ice. Use
the tight fitting pestle (Type A) to homogenize the sample for 30 strokes. Once finished,transfer the contents to a new 15 ml conical tube.
Monitor Cell Lysis: To ensure cell lysis, take 10 µl of the cell lysate from the dounce and lookat it under a phase contrast microscope using a hemocytometer to verify that the nuclei
have been released. It is often helpful to look at the cells before and after the lysis step as
this makes it easier to identify the nuclei versus whole cells. Intact cells should have a dark
central region (nucleus) surrounded by a halo of less dense cytoplasm. In lysed cells, thenuclei will appear as dots surrounded by asymmetric debris. If the cells are not lysed, thendounce on ice with an additional 10 strokes, or until the cells are lysed.

12.

Centrifuge for 3 minutes at 1,250 x g at 4°C.

13.

Remove the supernatant and discard. Resuspend each pellet in 500 µl - 1 ml ChIP Buffer
supplemented with PIC and 100 mM PMSF. (For 500 µl add 5 µl PIC and 5 µl PMSF. For 1 ml
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15

add 10 µl PIC and 10 µl PMSF.) Transfer the contents to a new 2 ml microcentrifuge tube.
14.

Incubate on ice for 10 minutes. Proceed to Section D: Chromatin Sonication of Tissue.

Section D. Chromatin Sonication of Tissue
The section below describes the fragmentation of chromatin using sonication. Due to theincreased concentration of protein and cellular debris present in animal tissue, we recommend
following this protocol for the preparation of chromatin and input DNA from tissue. Sonication results may vary depending on tissue type and sonication device being used. This protocol has beenvalidated using Active Motifs EpiShear™ Probe Sonicator in combination with the EpiShear™ Cooled
Sonication Platform to maintain probe height and temperature consistency between samples. We
do not recommend sonication of samples containing less than 50 mg tissue and/or 350 µl volume.
The ChIP Buffer has been optimized for immunoprecipitation performance, however, due to its
unique composition optimization of sonication conditions may be required. To maintain the highsensitivity of the assay, we recommend using our buffer system and altering the sonication time
and/or amplitude of your sonication system to achieve the desired fragmentation (e.g. somesystems may require as much as a three-fold increase in sonication time to improve chromatin
shearing). Please pay particular attention to our protocol regarding the processing of input chromatin for agarose gel analysis prior to the chromatin immunoprecipitation reaction as many steps
may differ from traditional ChIP protocols and failure to follow the outlined procedure may leadto artifacts in the gel images as shown in Figure 4 on page 18.
1.

Place the 2 ml microcentrifuge tube containing the chromatin into the tube cooler or packed
ice. Open cap and submerge the microtip into the liquid until the microtip is approximately
5 mm from the bottom of the tube. Sonicate according to optimized settings for the tissue
type being used (see Recommendations on page 10). A recommended starting range fortissue samples is: 25% amplitude, pulse for 30 seconds on and 30 seconds off for a total
sonication “on” time of 10 minutes (or 20 minutes elapsed time).

2.

Spin tubes at 4°C in a microcentrifuge at maximum speed for 2 minutes to pellet the cellulardebris.

3.

Transfer 25 µl of each chromatin preparation into a 250 µl PCR tube for analysis of shearing
efficiency and chromatin quantification.

4.

Aliquot the remainder of each chromatin preparation into 1.5 ml microcentrifuge tubes. We
recommend making aliquots of 150 µl volume and storing at -80°C.
Note:

The size of the chromatin sonication should be verified before proceeding to theimmunoprecipitation step.

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16

Chromatin Quantification
5. To each 25 µl chromatin preparation from Step 3 above, add 175 µl TE pH 8.0 and
2 µl RNAse A. Cap the PCR tubes and vortex to mix.
6.

Incubate in a thermocycler at 37°C for 1 hour.

7.

Add 5 µl Proteinase K to each tube, vortex and incubate in a thermocycler at 37°C for 3 hours.

8.

Add 10 µl 5 M NaCl, vortex and incubate at 65°C for 6-16 hours to reverse cross-links.

9.

Remove tubes from the thermocycler and add 250 µl phenol and 125 µl chloroform:isoamyl
alcohol (24:1). Vortex vigorously and spin tubes in a room temperature microcentrifuge at
maximum speed for 2 minutes.

10. Transfer each upper aqueous layer to a new 1.5 ml microcentrifuge tube and add 250 µl
chloroform:isoamyl alcohol (24:1). Vortex vigorously and spin tubes in a room temperature
microcentrifuge at maximum speed for 2 minutes.
11.

Transfer the upper aqueous layer to a new 1.5 ml microcentrifuge tube. Add 83 µl Precipitation Buffer, 2 µl Carrier and 900 µl absolute ethanol. Vortex to mix and chill at -80°C for 30
minutes to overnight.

12.

Spin at 4°C in a microcentrifuge at maximum speed for 15 minutes.

13.

Carefully remove the supernatant taking care not to disturb the pellet. Wash the pellet with
500 µl 70% ethanol and spin at 4°C in a microcentrifuge at maximum speed for 5 minutes.

14.

Carefully remove the supernatant taking care not to disturb the pellet. Remove residual
ethanol with a pipet tip. Leave the tubes uncapped and air dry for 10-15 minutes.

15.

When the pellets are dry, add 25 µl DNA Purification Elution Buffer to each tube. Incubate atroom temperature for 10 minutes. Then vortex to ensure the pellet is completely resuspended. This solution contains your DNA for quantification.

16.

Read the absorbance of each sample on a NanoDrop or other spectrophotometer at
260 nm to determine the DNA concentration of each chromatin preparation. Set aside
500 ng of DNA for analysis as described in Step 17. Store the remaining DNA at -20°C.

17.

Analyze each chromatin preparation on an agarose gel by following the instructions below.
a.

Prepare 500 mM NaCl by adding 2 µl 5M NaCl to 18 µl sterile water. Vortex to mix.

b.

Transfer 500 ng of Input DNA to a 250 µl PCR tube and add 1 µl 500 mM NaCl. Adjust
the final volume to between 10 µl with sterile water if needed.

c.

Heat samples in a thermocycler at 100°C for 20 minutes followed by ramping the temperature down to 50°C.

d.

Remove tubes from the thermocycler and incubate at room temperature for 5 minutes.

e.

Add gel loading buffer to each sample and run on a 1.5% agarose gel. Include 100 bp
and 1 kb DNA ladders to analyze chromatin size. DNA should appear as a smear anywhere between 200-1000 bp.

Note:

Chromatin may look different on an agarose gel than chromatin prepared using
traditional ChIP methods. However, this will not affect the sensitivity of the as-www.activemotif.com

17

say or increase background signal. Please follow the protocol as listed above forpreparing Input DNA. Use of an alternative reverse cross-linking method or omitting the 20 minute incubation at 100°C in NaCl is not recommended as this willcause artifacts that make the DNA appear larger. As long as the chromatin falls
within the recommended 200-1000 bp range, proceed with the ChIP reaction. If
fragments do not fall within this range sonication conditions should be further
optimized.
18. If chromatin preparations were successful, the aliquots stored at -80°C from Section D,
Step 4 can be used to perform the ChIP reactions in Section E.

Figure 4: Validation of chromatin shearing efficiency following reversal of cross-links overnight at 65°C.
Chromatin preparations were fixed and sonicated using the EpiShear™ Probe Sonicator and EpiShear™ Cooled Sonication Platform from Active Motif. Input DNA was prepared in duplicate according to Section D, Steps 5-16 in the manual.
In Sample 1, Step 17 was not performed and 500 ng of input DNA was loaded directly onto a 1.5% agarose gel without
receiving the addition of NaCl and incubation at 100°C. The omission of Step 17 has caused a buffer artifact that makes
the DNA appear larger on a gel. The duplicate sample, Sample 2, was processed according to the manual instructions andincluded the addition of NaCl and incubation at 100°C as stated in Step 17. Analysis of 500 ng of this input DNA on a 1.5%
agarose gel shows the expected fragmentation between 200-1000 bp. The difference in DNA sizing on the gel between
the two samples illustrates the importance of following the protocol recommendations regarding the processing of input
chromatin for agarose gel analysis prior to chromatin immunoprecipitation. Omission of key steps can lead to inaccurate
analysis of chromatin shearing efficiency. If the protocol steps were followed and the DNA fragments fall outside of therecommended range, sonication conditions should be further optimized.

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18

Protocols – Chromatin Immunoprecipitation
Section E. Immunoprecipitation
Successful chromatin immunoprecipitation depends on the quality of the ChIP antibody and theabundance of the target protein. Completion of this section requires a TAM-ChIP anti-species
secondary antibody conjugate (Catalog Nos. 53126 or 53127). Each TAM-ChIP antibody conjugate
is provided with the necessary pMENTS, oligonucleotide, TsTn5 transposase and 5X Tagmentationbuffer required to perform TAM-ChIP.
1.

Thaw sonicated chromatin on ice. Spin chromatin at 4°C in a microcentrifuge at maximum
speed for 2 minutes. Use the DNA concentration that was determined for your sonicatedchromatin sample to calculate the volume to use. We recommend using 5-10 µg chromatin(0.75 - 1.5 million cell equivalents) per ChIP reaction.

2.

Determine the number of ChIP reactions to perform. We strongly recommend setting up
a ChIP reaction without primary antibody to serve as a tagmentation control. This control
will show the effects of the transposase in the absence of genomic targeting and provide
a background reference during sequencing. Set up the ChIP reactions using separate 1.5 ml
microcentrifuge tubes for each ChIP antibody. Add the components in the order shown in
Table 1 below:
Table 1
Reagent

1 reaction

Tagmentation Control

Blocker

5 µl

5 µl

ChIP-validated antibody

4 µg

–

Sheared Chromatin

5-10 µg

5-10 µg

Protease Inhibitor Cocktail (PIC)

5 µl

5 µl

Adjust to 200 µl

Adjust to 200 µl

ChIP Buffer without SDS

3.

Cap tubes and incubate on an end-to-end rotator overnight at 4°C.

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19

4.

The next day, prepare the TAM-ChIP antibody conjugate and the oligo assembly in parallel.
Label separate microcentrifuge tubes for antibody conjugate and oligo assembly. If preparing
multiple ChIP reactions scale up the volumes accordingly. Refer to the lot-specific data sheet
provided with the TAM-ChIP anti-species conjugate for details on the volumes to use.

TAM-ChIP Antibody Conjugate

Oligo Assembly

Reagent

1 reaction

Reagent

1 reaction

Antibody conjugate

See lot-specificdata sheet

Oligonucleotide

See lot-specificdata sheet

pMENTsSee lot-specificdata sheet

pMENTsSee lot-specificdata sheet

Incubate for 30 minutes at RT
TsTn5 Transposase

Incubate for 30 minutes at RT

See lot-specificdata sheet

TsTn5 Transposase

Incubate for 1 hour at RT

See lot-specificdata sheet

Incubate for 1 hour at RT(Keep at RT until ready for use in Step 19)

5.

Remove the immunoprecipitation reaction from the rotator and quick spin to collect contents to the bottom of the tube. Add the assembled TAM-ChIP antibody conjugate to theimmunoprecipitation reaction. Pipet up and down to mix.

6.

Incubate for 1 hour at 4°C on an end-to-end rotator.

7.

Add 1 µl of 70 mM DTT to each sample. Pipet up and down to mix.

8.

Incubate samples at 37°C for 1 hour on an end-to-end rotator. During the incubation, prepare
the Protein G agarose beads as described in Step 9 below.

9.

The Protein G agarose beads require washing before use. Transfer 30 µl Protein G agarose
beads for each immunoprecipitation reaction to a 1.5 ml microcentrifuge tube. Add an equalvolume of TE, pH 8.0 and invert to mix. Spin at 1250 x g in a microcentrifuge for 1 minute.
Remove the supernatant equivalent to the volume of TE added to the agarose beads.
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20

Note:

Before pipetting the Protein G agarose beads, they should be fully resuspended
by inverting the tube. When pipetting the beads, cut 2 mm from the end of apipet tip to prevent the tip from becoming clogged.

10. Wash the beads a second time with the same volume of TE, pH 8.0. Invert to mix. Spin at
1250 x g for 1 minute in a microcentrifuge. Remove the supernatant equivalent to the volume
of TE added to the agarose beads. The beads are now ready to use.
11.

Spin the ChIP reactions at 1250 x g for 1 minute to collect liquid from the inside of the cap.

12.

Using a cut pipet tip, add 30 µl washed Protein G agarose beads to each immunoprecipitation
reaction. Cap tubes and incubate on an end-to-end rotator at 4°C for 2 hours.

13.

Label a ChIP Filtration Column for each ChIP reaction. Remove the tab from the bottom of
the column and place in an empty 1 ml pipet tip box as a holder (see Figure 5 on page 22).

14.

Remove ChIP reactions from rotator and spin at 1250 x g for 1 minute to collect liquid frominside of the cap.

15.

Transfer the entire ChIP reaction (including the protein G agarose beads) to the labeled ChIP
Filtration Column an allow flow-through to occur by gravity.

16.

Dry the bottom of each column with a Kim wipe and add a yellow plug to the bottom of the
ChIP filtration column to seal.

17.

Add 800 µl ChIP Buffer to each column and incubate at room temperature for 5 minutes.
Then, remove and discard the yellow plug. Allow flow-through to occur by gravity.

18. Once all the liquid has flowed through the column, blot the bottom of the column with a
Kim wipe and add a new yellow plug to the bottom of the ChIP filtration column.
19.

Prepare a Tagmentation Master Mix. Using the volumes required per reaction, calculate thevolumes needed for the master mix. Combine the reagents in the order shown in Table 2 to a
2 ml (or larger) tube. Pipette up and down to mix.
Table 2: Tagmentation Master Mix
Reagent

1 reaction

Master Mix

ChIP Buffer without SDS

400 µl X # rxns =µl

Tagmentation Buffer

100 µl X # rxns =µl

Oligo Assembly (From Step E.4)

variable X # rxns =µl

70 mM DTT

2.5 µl X # rxns =µl

Total Volume		variable

20. Calculate the single reaction total volume of tagmentation master mix needed. Activate
the TsTn5 transposase by adding the calculated volume of tagmentation master mix to eachlabeled ChIP Filtration Column.
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21

21.

Add a top cap to the tubes. We also suggest to seal the cap and plug with Parafilm to prevent any loss of liquid. Incubate reactions on an end-to-end rotator at 37°C for 1 hour.

22. At the end of the incubation, prepare the Elution Buffer. For each ChIP reaction, combine
90 µl of Elution Buffer AM4 and 10 µl 10X Elution Buffer AM2 in a 1.5 ml microcentrifuge tube.
Place at 37°C to pre-warm during the wash steps.
23. Remove ChIP reactions from rotator and discard the cap and plug. Allow flow-through tooccur by gravity.
24. Wash each column with 900 µl Wash Buffer AM1.
25. Repeat Step 24 four more times for a total of 5 washes.
26. Transfer columns to a new 1.5 ml microcentrifuge tube and spin in a room temperature
microcentrifuge at 1250 x g for 3 minutes to remove residual Wash Buffer.
27. Following the spin, transfer the ChIP Filtration Columns to new 1.5 ml microcentrifuge tubes.
Add 50 µl 37°C Elution Buffer to each column. Incubate at room temperature for5 minutes. Spin in a room temperature microcentrifuge at 1250 x g for 5 minutes.
28. With columns remaining in the same microcentrifuge tube, add another 50 µl 37°C Elution
Buffer to each column. Incubate at room temperature for 5 minutes and spin in a room
temperature microcentrifuge at 1250 x g for 5 minutes.
29. Discard the ChIP Filtration Columns. The flow-through (~100 µl volume) contains the ChIP
DNA. Proceed to Section F: DNA purification.

Figure 5: Using the ChIP Filtration Columns.
Remove the tab from the bottom of the ChIP Filtration Columns and place columns in an empty 1 ml pipet tip box toperform the wash steps.

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22

Protocols – ChIP DNA Purification
Section F. Reversal of Cross-links and DNA Purification
1.

Transfer each eluted ChIP DNA to a 250 µl PCR tube and add 2 µl Proteinase K. Vortex to mix
and heat in a thermocycler at 55°C for 30 minutes and then increase the temperature to 80°C
for 2 hours.

2.

To each PCR tube, add 150 µl phenol.

3.

Transfer reactions to Phase Lock Gel™ Heavy tubes (VWR, Catalog No. 10847-802).

4.

Add 75 µl chloroform:isoamyl alcohol and mix.

5.

Centrifuge for 5 minutes at maximum speed.

6.

Purify the DNA using Qiagen’s QIAquick PCR Purification Kit (Qiagen, Catalog No. 28104)
Note:

For column purification, add 5 volumes of the appropriate binding buffer to eachPhase Lock tube. Transfer each supernatant to a new tube. Then proceed withthe column purification protocol.

7.

Elute in 25 µl DNA Purification Elution Buffer or Qiagen EB Buffer.

8.

Purified DNA may be stored at -20°C for future use.

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23

Protocols – Library Amplification & Size Selection
Section G. Library Amplification
This section is designed to amplify the ChIP library using the DNA oligonucleotides that wereinserted into the chromatin by the TsTn5 transposase following genomic targeting. Completion ofthis section requires a TAM-ChIP Index and the Amplification primer mix provided with the antispecies TAM-ChIP conjugate (Catalog Nos. 53126 or 53127).
1.

Place a new 0.2 ml PCR tube on ice and prepare the library PCR reaction by adding components in the order shown in Table 3 below:
Table 3
Reagent

1 reaction

ChIP DNA

20 µl

TAM-ChIP i7 Index* (0.5 µM)

5 µl

Amplification primer mix (10 µM ea)

5 µl

5X Q5 Reaction Buffer

10 µl

Q5 High-Fidelity Polymerase (2 U/µl)

0.5 µldNTPs (10 mM)

1 µl

Sterile H2O

8.5 µlTotal volume

50 µl

Note:

If combining multiple TAM-ChIP samples together for sequencing, make sure touse unique TAM-ChIP Index primers for each sample.

2.

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

4.

Place in the thermal cycler and run the library PCR thermal cycler program as described
below:
Library PCR
Thermal Cycler
Program

98°C for 30 seconds
63°C for 3 minutes
98°C for 10 seconds
63°C for 30 seconds x 14 cycles
72°C for 30 seconds
4°C Hold

Proceed immediately to Library Size Selectionwww.activemotif.com

24Section H: Library Size Selection
This section is designed to clean up the Library PCR reaction using AMPure beads and freshly
prepared 80% ethanol. Libraries should be between 200-500 bases.
1.

To each PCR reaction, add 50 µl ddH2O.

2.

Add 100 µl AMPure beads to each reaction. Pipet up and down repeatedly (~10 times) toensure a homogenous mixture. Incubate at room temperature for 5 minutes.

3.

Place the tubes on the magnet to separate. Let sit for 2 minutes. Remove and discard thesupernatant.

4.

Without removing the tubes from the magnet, add 200 µl 80% ethanol to wash the beads.
Allow beads to sit in the ethanol wash for 30 seconds before removing and discarding thesupernatant.

5.

Repeat Step 4 one more time.

6.

Without removing the tubes from the magnet, allow the beads to air dry for 5 minutes.

7.

Remove the tubes from the magnet and add 20 µl DNA Purification Elution Buffer to thetube. Pipet up and down to mix.

8.

Place the tubes on the magnetic stand to separate. Transfer the supernatant to a new PCR
tube. This contains the eluted DNA library.

9.

Assess the DNA size using a TapeStation or equivalent method to ensure it falls within the200-500 base range.

10. The library can be quantified and is ready for sequencing on the Illumina platform.

Section I. Sequencing Guidelines
For sequencing, we recommend 30 million reads. For additional information regarding the run
set-up, sample sheet preparation, data analysis and de-duplication of the MIDs, please visit Active
Motif’s website www.activemotif.com/tamchip and download the Guideline for TAM-ChIP
Molecular Identifier (MID) Analysis from the Documents tab. Or contact Technical Support at
tech_service@activemotif.com

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25

Appendix
Section J. 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 formaldehyde fixation and centrifugation (intact cell pellet), -80°C.
2. After chromatin shearing, -80°C.
3. After DNA clean up, -20°C.

After sonication shearing
and centrifugation, aviscous or cloudy layer isvisible in the chromatin.

Depending upon the cell type, lipid or glycogen layers may be seen after centrifugation.
For example, fatty tissues may have a lipid layer. Avoid such layers when you remove thesupernatant. However, if the whole supernatant is cloudy, it should not interfere with the
IP reaction.

Poor yield of shearedchromatin.

Insufficient cell numbers were used. Repeat using a larger number of cells.
Nuclei not released. It is highly recommended to perform dounce homogenization, evenwhen using sonication. Use a dounce homogenizer with a small clearance pestle (Active
Motif Catalog Nos. 40401 & 40415). Monitor cell lysis under a microscope. Generally, themore cells that are lysed, the higher the sheared chromatin yield.
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.
Use fresh formaldehyde when preparing Complete Cell Fixation Solution and Complete
Tissue Fixation Solution.
Buffers were not scaled proportionally to the size of the sample. Use the chart in Cell
Growth Recommendations to scale up or down chromatin preparation.

Shearing efficiency is notclear from gel analysis.

Material is stuck in the wells, and smears or streaks are seen from the top to bottom of
the lane. The sheared chromatin needs to have the cross-links reversed, protein removed(Proteinase K) and RNA removed (RNase), followed by DNA purification.
High molecular weight products. Decrease the size of the fragments by re-sonicating thesample. If an alternative reverse cross-linking method was used, or the 20 minute incubation at 100°C in NaCl was omitted prior to running the agarose gel for analysis, please
repeat the input chromatin preparation and follow the manual instructions.

Performing ChIP with alarge volume of chromatin.

This is not recommended. It is better to set up several small ChIP reactions. Do not
perform a single scaled-up reaction, as the capture efficiency is lower.

High background.

Antibody issue. Too much antibody relative to the amount of chromatin in the ChIP
reaction. Excess antibody will result in more non-specific binding, which will be detected
as increased background.

Poor or no enrichmentwith target antibody.

Too little chromatin. Generally, we recommend using 5 - 10 µg of chromatin per ChIP reaction. Be sure to quantitate the concentration of the sheared chromatin sample(s) being
ChIP’d to ensure both that adequate chromatin is used per sample, and that equal massquantities of chromatin are used in each ChIP.
Antibody is not ChIP-Seq validated, or has low binding affinity. The antibody does notefficiently recognize fixed proteins, either because the epitope is destroyed by fixation or
because the epitope is masked by other proteins in a larger complex. To assist in ChIP-Seq
validating an antibody, it is very useful to use a positive control antibody such as Histone
H3K4me3 (Catalog No. 39915).
Wrong anti-species TAM-ChIP antibody conjugate was used. Ensure that the TAM-ChIP
conjugate will properly detect the species (rabbit or mouse) of your ChIP-Seq validated
primary antibody.

www.activemotif.com

26Section K. 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®		ChIP-IT® qPCR Analysis Kit		
Next Gen DNA Library Kit		
Next Gen Indexing Kit		
Low Cell ChIP-Seq		
ChIP-IT® ChIP-Seq		
ChIP-IT® FFPE		ChIP-IT® FFPE Chromatin Preparation Kit		
ChIP-IT® Express HT		
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
10 rxns
16 rxns
64 rxns
16 rxns
10 libraries
16 rxns
5 rxns96 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
53029
53216
53264
53084
53041
53045
53030
53018
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		

50 rnxs
25 rxns

54001
54002

Modified Histones Array		

FormatCatalog No.

MODified™ Histone Peptide Array		

1 array13001

www.activemotif.com

27

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
E-mail: tech_service@activemotif.com

Toll Free: 877 222 9543
Direct: 760 431 1263
Fax: 760 431 1351

Active Motif Europe
Avenue Reine Astrid, 92
B-1310 La Hulpe, Belgium
E-mail: eurotech@activemotif.com
Direct: +32 (0)2 653 0001

Germany Free Phone: 0800 181 99 10
France Free Phone: 0800 90 99 79
UK Free Phone: 0800 169 31 47
Fax: +32 (0)2 653 0050

Active Motif Japan
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2-21 Ageba-Cho, Shinjuku-Ku
Tokyo, 162-0824, Japan

Direct: +81 3 5225 3638
Fax: +81 3 5261 8733
E-mail: japantech@activemotif.com

Active Motif China
787 Kangqiao Road
Building 10, Suite 202
Pudong District
Shanghai, 201315, China

Direct: (86)-21-20926090
Hotline: 400-018-8123
E-mail: techchina@activemotif.com

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