enChIP®
(version B1)
Catalog No. 53125

Active Motif North America
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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
kind, either explicit or implied. Information may be changed or updated in this manual at any time.
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The manufacturer of this documentation is Active Motif, Inc.
© 2016 Active Motif, Inc., 1914 Palomar Oaks Way, Suite 150; Carlsbad, CA 92008. All rights reserved.
All trademarks, trade names, service marks or logos referenced herein belong to their respective
companies.
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TABLE OF CONTENTS

Page

Overview  . . . . . . .  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Introduction  . . . .  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2
References . . . . . .  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3
Flow Chart of Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Kit Performance and Benefits  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Kit Components and Storage
enChIP® Kit  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Additional Materials Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Protocols – Experimental Set Up
Design of gRNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Transfection of gRNA and dCas9 expression plasmids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Buffer Preparation & Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Protocols – Preparation of Sheared Chromatin
Section A. Cell Fixation Starting with Cultured Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Section B. Chromatin Sonication of Cultured Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  16
Protocols – Chromatin Immunoprecipitation
Section C. Protein G Magnetic Bead Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  19
Section D. Immunoprecipitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Section E. Washing of IP Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Section F. Elution, Reversal of Cross-links and DNA Purification . . . . . . . . . . . . . . . . . . . 22
Protocols – ChIP DNA Analysis
Section G. qPCR Primer Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Section H. Quantitative PCR (qPCR) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Section I. ChIP-Seq . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
Appendix
Section J. Troubleshooting Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
Section K. Related Products  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
Technical Services  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

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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 the identification of proteins bound to specific DNA
loci. With the advent of the CRISPR/Cas9 technology, new applications are being applied to study
chromatin interactions.
With enChIP®* (engineered DNA-binding molecule-mediated chromatin immunoprecipitation), the
CRISPR (clustered regularly interspaced short palindromic repeats) system is used to direct a guide
RNA (gRNA) to a specific genomic locus for immunoprecipitation. The gRNA is designed as thecomplementary sequence of the desired target locus. The gRNA is co-expressed with a deactivated (enzymatically inactive) form of the Streptococcus pyogenes Cas9 endonuclease (dCas9). The
dCas9 protein is expressed with Active Motif’s unique AM-tag sequence, which was specifically designed for use in ChIP. The AM-tag has minimal cross-reactivity with mammalian samples, thereby
reducing background signal. Another benefit of the AM-tag is that the sequence is unstructured
which allows the tag to protrude from the protein for maximum exposure during immunoprecipitation. Following transfection of the gRNA and AM-tagged dCas9, cells are formaldehyde fixed,
lysed and the chromatin is fragmented by sonication. An antibody directed against the AM-tag isused to enrich for genomic sequences bound by the gRNA/dCas9 complex. Following immunoprecipitation, DNA can be analyzed by qPCR or NGS to identify the enriched genomic regions. enChIP
provides the opportunity to investigate off-target effects of gRNA design, or to study cis- andtrans-interacting chromosomal looping events.
To use Active Motif’s enChIP® Kit simply clone your gRNA sequence into one of Active Motif’s
expression vectors. Following transfection and expression of the gRNA and AM-tagged dCas9, the
enChIP Kit can be used to isolate chromatin and perform immunoprecipitation using an antibody
directed against the AM-tag. The enChIP Kit contains sufficient reagents to perform 16 chromatin
preparation and immunoprecipitation reactions.
product

formatcatalog no.

enChIP® Kit

16 rxns53125

pAM_gRNA Vector

10 µg

53121

pAM_dCas9 Vector

10 µg

53122

pAM_gRNA_CTCF Vector

10 µg

53123

pAM_dCas9_CTCF Vector

10 µg

53124

FuGENE® HD Transfection Reagent

0.2 ml32042

AM-Tag polyclonal antibody

100 µg61677

ChIP-IT qPCR Analysis Kit

10 rxns

53029*Patent number: WO2014/125668; JP5954808
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1

Introduction
The CRISPR/Cas system can be traced to bacteria and archaea which use the system to target andinactivate foreign genetic material. This adaptive immunity mechanism relies on the acquisitionof spacers, the assembly of the CRISPR RNAs (crRNAs) and Cas proteins to the invading nucleic
acids and degradation of the foreign material by nucleases1. There are three main types of CRISPR/
Cas systems, each with a specific Cas protein. Type II systems utilize Cas9 complexes1. Recently,
researchers have repurposed the CRISPR/Cas9 system for genome engineering of mammaliansystems2-6.
In type II CRISPR/Cas systems, the crRNA hybridizes with a transactivating crRNA (tracrRNA) that
contains additional nucleotide sequences. In the presence of Cas9, the crRNA-tracrRNA hybrid
is processed to generate a spacer that is 20 nucleotides in length7,8. The Cas9 complex thenundergoes a conformational change for DNA binding1,4,9. The spacer sequence directs the Cas9
complex to a complementary DNA sequence containing a protospacer adjacent motif, or PAM
site (5´-NGG). Recognition of a PAM site leads to unwinding of the DNA and formation of an
RNA-DNA heteroduplex7,9,10. Nucleases are then activated to create a double-stranded break in thetarget DNA.
Engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) was firstdescribed in 2013 by Toshitsugu Fujita and Hodaka Fujii11, 12. enChIP was designed to purify specificgenomic regions using the CRISPR/Cas9 system. A single guide RNA vector containing a fusionof the crRNA-tracrRNA sequence (5´side to identify the DNA target sequence and a 3´duplex
RNA structure to bind Cas9) is used to clone the target sequence of interest. When the gRNA isexpressed in combination with a tagged deactivated Streptococcus pyogenes Cas9 (dCas9) protein, specific genomic regions can be immunoprecipitated using an antibody directed against thetag. By using a catalytically inactive form of Cas9 endonuclease, double-stranded breaks are notintroduced and DNA, RNA and proteins associated with the target sequence can be recovered11-15.

With Active Motif’s enChIP Kit, simply clone your 20 nucleotide target sequence into Active
Motif’s gRNA or dCas9 expression vectors. Following transfection and expression of the gRNA and
AM-tagged dCas9, cells are formaldehyde fixed, lysed and the chromatin is fragmented by sonication. An antibody directed against the AM-tag is used to enrich for genomic sequences bound by
the gRNA/dCas9 complex. Following immunoprecipitation, DNA can be analyzed by qPCR or NGS
to identify the enriched genomic regions.
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2For researchers looking to perform genome editing experiments, determining the specificity of
the gRNA binding site is critical. If the gRNA target sequence demonstrates off-target binding,then genome editing methods using active Cas9 protein could lead to unwanted downstream
effects. By using the enChIP Kit containing the dCas9 protein, off-target gRNA binding sites can be
identified to determine the quality of the gRNA design prior to use in genome editing experiments. Additionally, enChIP can be used to study cis- and trans- chromosomal interactions. Evaluation of the immunoprecipitated DNA sequences may reveal chromosomal looping events.

References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.

van der Oost, J. et al. (2014) Nat. Rev. Microbiol., 12(7): 479-492.
Cong, L. et al. (2013) Science, 339(6121): 819-823.
Mali, P. et al. (2013) Science, 339(6121): 823-826.
Hsu, P. et al. (2014) Cell, 157(6): 1262-1278.
Doudna, J.A. and Charpentier, E. (2014) Science, 346(6213): 1258096.
Harrison, M.M. et al. (2014) Genes Dev., 28(17): 1859-1872.
Deltcheva, E. et al. (2011) Nature, 471(7340): 602-607.
Jinek, M. et al. (2012) Science, 337(6096): 816-821.
Nishimasu, H. et al. (2014) Cell, 156(5): 935-949.
Sternberg, S.H. et al. (2014) Nature, 507(7490): 62-67.
Fujita, T. and Fujii, H. (2013) Biochem. Biophys. Res. Commun., 439(1): 132-136.
Fujita, T. et al. (2013) Sci. Rep., 3: 3171.
Fujita, T. and Fujii, H. (2014) PLoS ONE, 9(7): e103084.
Fujita, T. et al. (2015) PLoS ONE, 10(4): e0123387.
Fujii, H. and Fujita, T. (2015) Int. J. Mol. Sci., 16(9): 21802-21812.

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3Flow Chart of Process
Design and clone gRNA target sequence

pAM_dCas9

pAM_gRNA

pAM_dCas9

Dual vector transfection

Single vector transfection

Experimental
Set-Up

Day 1
Transfect expression vector(s) containing gRNA and AM-tagged dCas9 protein
Day 2
Passage cells

enChIP Kit

Day 3
Formaldehyde fix cells & prepare chromatin
Protein G Magnetic Bead Preparation& Immunoprecipitation

Day 4

Washes, elution, reversal of cross-links
and DNA purification

Day 5

qPCR Analysis

Library preparation and Next-Gen sequencing

Flow Chart of the enChIP method.
Prior to using the enChIP Kit (Catalog No. 53125), a guide RNA (gRNA) target sequence is selected to target a specificgenomic region. The gRNA sequence is then cloned into either the pAM_gRNA expression vector for dual vector transfections, or cloned into the pAM_dCas9 expression vector for single vector transfection. 24 hours post-transfection of thegRNA and AM-tagged dCas9 expression vectors, cells are passaged. Another 24 hours later cells are formaldehyde fixed andchromatin is prepared. Protein G magnetic beads are blocked and the chromatin is pre-cleared. An overnight immunoprecipitation using the AM-Tag monoclonal antibody captures the gRNA/Cas9 genomic complexes. Following washes, the DNA
is eluted from the beads, the cross-links are reversed and the enriched DNA is purified. Recovered DNA can be analyzed by
qPCR or Next-Generation sequencing.
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4Kit Performance and Benefits
enChIP Kit Applications:• Identify off-target binding events of the gRNA
• Evaluate cis- and trans-interacting chromosomal looping• Works well with open chromatin regions to detect promoter, enhancer and insulator elements. enChIP is not optimized for use on repressive regions.

Figure 1: Comparison of enChIP performance to evaluate different genomic loci.
To evaluate different genomic loci, gRNAs were designed to target known insulator, promoter, enhancer and repressive
regions of the genome. Experiments were run according to the instructions in the enChIP Kit. ChIP-seq results show highly
specific enrichment of the insulator, promoter and enhancer regions. For the repressive genomic region, only a small ChIPseq peak was observed indicating that the enChIP method is not optimized for repressive regions.

Figure 2: Determining gRNA target sequence specificity.
Four different gRNAs were designed using the CRISPRdirect program to target a 200 bp region surrounding a CTCF bindingsite on chromosome 19. Following transfection of the gRNA and dCas9 into HEK293T cells, chromatin was prepared andimmunoprecipitated according to the instructions in the enChIP Kit. enChIP-seq results show the differences in gRNA
specificity. gRNA 2 has a high degree of background as compared to gRNA 4 despite having only a single bp difference inthe target sequence. This confirms the value of performing enChIP to identify off-target binding events of the gRNA prior
to use in genome engineering experiments.
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5Kit Components and Storage
Please store each component at the temperature indicated in the table below. Do not re-freeze
the Protein G Magnetic Beads. Once thawed, Protein G beads should be stored at 4°C.
Reagents

Quantity

Storage

AM-Tag monoclonal Ab (1 mg/ml)

80 µg

-20°C

RNase A (10 µg/µl)

40 µl

-20°C

Proteinase K (10 µg/µl)

80 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

Anti-mouse IgG (1 mg/ml)

60 µg

4°C

Fixation Buffer

2 x 1.5 ml

4°C

Protein G Magnetic Beads*

1.5 ml4°C

Detergent

25 ml

RT

Stop Solution

20 ml

RT

Chromatin Prep Buffer

85 ml

RT

ChIP Buffer

35 ml

RT

Equilibration Buffer

2 x 120 ml

RT

Blocking Buffer AM4

15 ml

RT

LS Buffer

35 ml

RT

Wash Buffer AM1

100 mlRT

LiCl Buffer

35 ml

RT

Elution Buffer AM4

2 x 1.5 ml

RT

TE

50 ml

RT

DNA Purification Binding Buffer

50 ml

RT

3 M Sodium Acetate

500 µlRT

DNA Purification Wash Buffer**

10 ml

RT

DNA Purification Elution Buffer

5 ml

RT

DNA Purification Columns

16 ea

RT

* The Protein G Magnetic Beads are shipped on dry ice and can be stored frozen until their first
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6

use. Once thawed, the Protein G beads should not be re-frozen by the customer. Protein G
Magnetic Beads should be stored at 4ºC.
**Requires the addition of ethanol before use.
Additional materials required for enChIP Kit• Transfected cultured cells containing a sequence verified gRNA target in combination withthe pAM_dCas9 expression vector and a no gRNA control transfection. Refer to Experimental Set-Up on pages 8-13 to design, clone and transfect cells.
• 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 for shearing
chromatin.
• 37% formaldehyde solution with 10-15% methyl alcohol to prevent polymerization (e.g. Sigma
Aldrich Catalog No. 252549). Do not use paraformaldehyde.
• 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))• Pipettors and tips (filter tips are recommended)• Rocking platform for culture plates• Cell scraper (rubber policeman)• 15 and 50 ml conical tubes• 100% ethanol (absolute)• 70% ethanol• DNase-free H2O• 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• 0.2 ml PCR tubes• Thermocycler• Spectrophotometer for DNA quantitation• Agarose gel electrophoresis apparatus• (Optional) ChIP-IT® qPCR Analysis Kit (Catalog No. 53029)• (Optional) Gene-specific qPCR primer pairs for enrichment analysis• (Optional) SYBR Green qPCR master mix (Bio-Rad Catalog No. 170-8882)

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7

Protocols – Experimental Set Up
PLEASE READ THE ENTIRE PROTOCOL BEFORE STARTING!
Design of gRNAIn the enChIP Kit, a guide RNA (gRNA) is designed to target a specific genomic locus. Online software programs are available to assist in the design of a 23 bp gRNA in the form of 5´-N20NGG-3´,
where the NGG represents the Protospacer Adjacent Motif (PAM) sequence immediately following
the target sequence that is required for successful binding of Cas9 protein. The identified gRNAsites may reside on the + or - strand. We recommend designing multiple gRNAs to a specific locus
and then experimentally testing each gRNA for specificity using the enChIP Kit. If possible, try to
avoid target sequences that overlap with known recognition sites for DNA-binding proteins as thismay block access of the gRNA to its target location.
1.

Determine the genomic region of interest for gRNA targeting. Retrieve the DNA sequenceof approximately 500 bp surrounding this target locus. This sequence will be analyzed using
online software programs and gRNA selection tools to identify the best gRNA sequences.

2.

There are several online programs available to assist with gRNA design. A few are listed
below. Select a program and follow the software provider’s instructions for use.
CRISPRdirect - http://crispr.dbcls.jp
Michael Boutros Lab - http://www.e-crisp.org/E-CRISP/designcrispr.html
Feng Zhang Lab - http://crispr.mit.edu

3.

Below is an example of gRNA design using CRISPRdirect. Software instructions may changeover time, so please follow the recommendations of the software provider for use.
a.

Paste the genomic coordinates or genomic sequence from Step 1 above into the
CRISPRdirect site.

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8b.

Check the box for “show highly-specific targets only”.

c.

The output box will show potential gRNA sequencing for the genomic target.

d.

Click the “gRNA” link to the right of the sequence and copy the 20 bp non-PAM
sequence.

e.

Select the transfection method (dual vector or single vector) desired to perform yourexperiment. Based on this decision, paste the 20 bp non-PAM sequence from step 3dabove into the correct gRNA insert template. gRNA templates can be found on page 10
and downloadable versions are available online at www.activemotif.com/enchip. Please
note that each cloning vector has unique overlapping ends and gRNA inserts are notinterchangeable between the different vectors.
Dual Vector Transfection: The gRNA is cloned into a vector designed specifically to
express the gRNA. The dCas9 protein is expressed in a separate expression vector. This
option requires co-transfection of two vectors (gRNA vector + dCas9 vector) into yourcell line of interest. Perform cloning using the pAM_gRNA vector (Catalog No. 53121).
Single Vector Transfection: The gRNA sequence of interest is cloned directly into thedCas9 expression vector. Both the gRNA and the dCas9 protein will be expressed fromthe single vector. This option requires only a single transfection, but the vector size
is extremely large (>10 kb) and may prove challenging to transfect in some cell lines.
Perform cloning using the pAM_dCas9 vector (Catalog No. 53122).
Using existing gRNA vectors: For researchers who already have their gRNA of interest
cloned into an expression vector, the dual vector transfection system should be used.
This involves co-transfection of the researchers’ gRNA expression vector with Active
Motif’s pAM_dCas9 vector (Catalog No. 53122). No additional cloning is required.
Proceed to the next section for Transfection of gRNA and dCas9 expression plasmids.

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9

Figure 3: gRNA insert templates should be used for the design of gBlocks® Gene Fragments for cloning into theappropriate expression vector for either single or dual vector transfections. Color images for the gRNA insert
templates can be found online at www.activemotif.com/enchip.

4.

Synthesize gBlocks® Gene Fragments (Integrated DNA Technologies, https://www.idtdna.
com/site/order/gblockentry) representing the entire 453 bp sequence to be cloned into
the gRNA expression plasmid. The gBlocks sequence should contain the overlapping ends
needed for subcloning into the expression vector of choice, the 20 bp target sequence, theguide RNA scaffold and the termination signal.

5.

Linearize the appropriate expression plasmid using restriction enzyme digestion.
a.

pAM_gRNA vector: Linearize the pAM_gRNA vector with a double digest using
restriction enzymes SpeI and HindIII. Gel purify the 3891 bp digested backbone.

b.

pAM_dCas9 vector: Linearize the pAM_dCas9 vector with a single digest using
restriction enzyme MfeI.

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10

6.

Clone each gBlocks gRNA insert into Active Motif’s expression plasmid using directional cloning methods such as InFusion® (Clontech) or Gibson Assembly® (New England Biolabs). Follow
the manufacturer’s recommendations for cloning.
a.

7.

For InFusion cloning, we recommend a 1:3 molar ratio of vector : insert. For example:
Use 150 ng pAM_gRNA vector with 50 ng gRNA insert.
Use 150 ng pAM_dCas9 vector with 22 ng gRNA insert.

Perform transformations of each gRNA construct into competent E.coli. We recommend theuse of Active Motif’s RapidTrans TAM1 Competent cells. (Catalog No. 11096). Plate transformation reactions onto LB agar plates containing the appropriate antibiotic selection for thecloning vector used. Grow overnight at 37°C.
pAM_gRNA vector: Kanamycin selection is required
pAM_dCas9 vector: Ampicillin selection is recommended

8.

Select 2-4 colonies for each gRNA construct and isolate the plasmid DNA. The DNA shouldbe free of nucleases or other contaminants prior to transfection. Sequence validate eachplasmid to confirm the correct sequence. We suggest preparing a glycerol stock for eachcandidate construct.

9.

Proceed to the next section for Transfection of gRNA and dCas9 expression plasmids.

Transfection of gRNA and dCas9 expression plasmids
Following sequence validation of the expression constructs, cells can be transiently transfectedwith the gRNA and pAM_dCas9 expression plasmids. The following protocol provides recommendations for transfection using FuGENE® HD Transfection Reagent (Catalog No. 32042). Optimization
may be required for each cell line and expression construct tested.
To determine the efficiency of the transfection, set up a duplicate transfection for each cell typeused. Cell lysates can be prepared from the duplicate transfection reaction using Active Motif’s
Nuclear Extract Kit (Catalog No. 40010) for analysis by Western blot. Use the AM-Tag polyclonal
antibody (Catalog No. 61677) at a 1:250 - 1:1,000 dilution for detection of the AM-tagged dCas9
protein. If the tagged protein is not detected, continue to optimize transfection conditions.
Calculate the number of transfections you will perform with each gRNA and cell line. Small scale
reactions provide enough chromatin to perform one enChIP reaction. Large scale reactions provide
enough chromatin to perform 5 enChIP reactions. We recommend running a no gRNA negative
control reaction for each cell line tested. Active Motif also offers positive control vectors for bothdual or single transfection. The positive control gRNA targets a locus corresponding to a CTCF
binding site on chromosome 19 (5,804,115 – 5,804,290). These vectors are available as pAM_gRNA_
CTCF vector (Catalog No. 53123) and pAM_dCas9_CTCF vector (Catalog No. 53124).

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11

1.

Seed cells in either a 6-well plate (small scale) or 10cm dish (large scale) using the appropriate
growth medium. Incubate in a humidified incubator for 24 hours. Cells should be 70-80%
confluent at the time of transfection.
Small Scale Cell Culture(1 enChIP rxn)
Single Vector

Dual Vector

Large Scale Cell Culture(5 enChIP rxns)
Single Vector

Dual Vector

Cell culture plate

6-well plate

6-well plate

10 cm dish10 cm dishCell seeding density•

5 x 105 cells

5 x 105 cells

3 x 106 cells

3 x 106 cells

Growth medium

2 ml

2 ml

10 ml

10 ml

*These conditions were established for cells with doubling times of 14-18 hours. Cell seeding densities may need to
be optimized to ensure that cells are ~80% confluent at the time of transfection.

2.

Prepare a separate microcentrifuge tube for each transfection reaction. To each tube add therecommended amount of DNA and Opti-MEM according to the table below.

Test gRNAor
Positive control
CTCF gRNA

No gRNA
negative control

Small Scale Cell Culture

Large Scale Cell Culture

SingleVectorDual
VectorSingleVectorDual
VectorgRNA vector

–

1 µg

–

2 µg

dCas9 vector

–

1 µg

–

2 µg

(dCas9 + gRNA) vector

1 µg

–

2 µg

–

Opti-MEM

Up to 275 µl

Up to 275 µl

Up to 550 µl Up to 550 µl

gRNA vector

–

–

–

–

dCas9 vector

1 µg

1 µg

2 µg

2 µg

–

–

(dCas9 + gRNA) vector––

Opti-MEM

Up to 275 µl

Up to 275 µl

Up to 550 µl Up to 550 µl3.

Add FuGENE HD Transfection reagent drop wise directly to the DNA/media mixture. Do notallow FuGENE to come directly in contact with the plastic from the tube. Mix the solution by
pipetting up and down and incubate at room temperature for 30 minutes.
Small scale cell culture: Add 6 µl FuGENE to each transfection reaction.
Large scale cell culture: Add 12 µl FuGENE to each transfection reaction.

4.

Add the entire DNA/media/FuGENE mixture drop wise to each cell culture plate. Incubate
on a shaking platform at 100 rpm for 2 minutes to evenly distribute the transfection mixture.

5.

Return plate to humidified incubator for 24 hours.

6.

24 hours post-transfection, passage cells to a larger dish. Add additional complete growth
medium to the cells.
Small scale cell culture: Transfer cells in each well of a 6-well plate to a 10 cm dish.
Culture cells in 10 ml complete growth medium.
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Large scale cell culture: Transfer cells from a 10 cm dish to a 15 cm dish. Culture cells in
20 ml complete growth medium.
7.

Return plate to humidified incubator for 24 hours.

8.

Transfected cells are now ready to be processed for chromatin fixation. Alternatively, if
duplicate reactions were performed cell lysates can be prepared using Active Motif’s Nuclear
Extraction Kit (Catalog No. 40010) for Western blot analysis of transfection efficiency using
the AM-Tag polyclonal antibody (Catalog No. 61677) to recognize the tagged dCas9 protein.

Buffer Preparation & Recommendations
Prepare the following buffers for use in chromatin preparation and immunoprecipitation reactions.
All other buffers and reagents are supplied ready for use.
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, lab
coat and working in a ventilated hood), add 750 µl 37% formaldehyde to the tube and vortex tomix. 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.
Stop Solution
Is provided ready to use. Use 1/20 media volume per cell culture plate.
PBS Wash Buffer
Prepare 25 ml PBS Wash Buffer for every 15 cm plate. To a 50 ml conical tube add 21.25 ml sterilewater, 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. Use appropriate safety precautions (i.e. safety glasses, gloves and lab coat) when working with PMSF as it is highly toxic.
Protein G Magnetic Beads
The supplied magnetic beads require processing prior to use. Follow the instructions in the
manual to prepare the beads for use in the enChIP reactions. For best results, gently shake andinvert the tube to resuspend the magnetic beads. The beads settle quickly, and therefore should
be resuspended just before pipetting. Protein G Magnetic Beads are shipped on dry ice andcan be stored frozen until their first use. Once thawed, beads should not be re-frozen by thecustomer. Protein G Magnetic Beads should be stored at 4ºC.

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ChIP Buffer, LS Buffer, Wash Buffer AM1, LiCl Buffer and TE
These buffers are provided at room temperature. We recommend using chilled buffers for the
immunoprecipitation set-up and wash steps. Place these buffers on ice prior to use.
AM-Tag Monoclonal Antibody
We recommend using 5 µg AM-Tag monoclonal antibody per enChIP reaction. The AM-Tag monoclonal antibody is provided at a concentration of 1 µg/µl, so you will need to use 5 µl per enChIP.
Anti-Mouse IgG
The anti-mouse IgG is provided at a concentration of 1 µg/µl, so you will need to use 3.5 µl per
chromatin pre-clearing reaction.
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 the enChIP Kit. Indirect sonication systems
may require longer sonication times to achieve optimal chromatin shearing. enChIP experiments
usually require chromatin that has been sheared to a size of 200-2000 bp. In general, shearing efficiency is improved through the use of a small shearing volume and a V-bottom tube rather than around-bottom tube. Also, note that shearing is inefficient if the chromatin sample becomes emulsified with air 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 toremove trapped air. Then, resuspend any pelleted material and transfer chromatin to a new tube
before continuing sonication. 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.
DNA Purification Wash Buffer
The DNA Purification Wash Buffer requires the addition of ethanol before use. The final concentration of ethanol should be 80%. Add 40 ml of fresh 100% ethanol to the DNA Purification Wash
Buffer bottle. Invert repeatedly. The buffer can be stored at room temperature after the additionof ethanol. The ethanol only needs to be added before the first use, after that the Wash Buffer isready for use.
3M Sodium Acetate
It is important to check the sodium acetate before use to ensure that the salts have not precipitated out of solution. Once the sodium acetate is in solution it should be stored at room
temperature.
.
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Protocols – Preparation of Sheared Chromatin
Section A: Cell Fixation of Cultured Cells
This protocol describes cell fixation and chromatin preparation using the transfected cell lines.

Cell Fixative Solution

10 cm
dish

15 cm
dish

1 ml

2 ml

Stop Solution

550 µl1.1 mlPBS Wash Buffer

2 x 5 ml2 x 10 ml

Chromatin Prep Buffer

5 ml

5 ml

ChIP Buffer

Up to 500 µl

Up to 1 ml

1.

Prepare Complete Cell Fixation Buffer as described on page 13. 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). The cell fixation solution can be added to the growth medium in the presence orabsence of serum.

2.

Shake gently at room temperature for 10 minutes.

3.

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).

4.

Swirl to 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 5-10 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 5-10 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
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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.

Prepare Complete ChIP Buffer by adding 10 µl PIC and 10 µl PMSF per 1 ml ChIP Buffer.

13.

Remove the supernatant and discard. Resuspend each nuclear pellet in Complete ChIP Buffer.
Small scale cell culture: Resuspend each pellet to reach a final volume of 500 µl.
Large scale cell culture: Resuspend each pellet to reach a final volume of 1 ml

14.

Transfer the contents to a new 2 ml microcentrifuge tube.

15.

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.
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. some systems may require as much as a three-fold increase in sonication time to improve chromatin shearing). We recommend checking shearing efficiency prior to performing the immunoprecipitation
reactions. Input DNA can be prepared and run on an agarose gel to confirm shearing efficiency.
The chromatin should appear as a smear between 200-2000 bp.
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 14). Suggestions for sonication conditions
are provided below based on the chromatin volume:
For 500 µl chromatin volumes: 42% amplitude, pulse for 30 seconds on and 30 seconds off
for a total sonication “on” time of 10 minutes (or 20 minutes elapsed time).
For 1 ml chromatin volumes: 61% amplitude, pulse for 30 seconds on and 30 seconds off fora 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. This is the Input DNA.
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4.

The remaining chromatin should be stored at -80°C.
For 500 µl chromatin volumes: The entire amount will be used in the enChIP reaction. Thereis no need to aliquot.
For 1 ml chromatin volumes: Use 200 µl per enChIP reaction. Prepare 200 µl aliquots into
1.5 ml microcentrifuge tubes.

Input Preparation
5. To each 25 µl chromatin preparation from Step 3 above, add 175 µl TE 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 Input DNA.

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 Input DNA at -20°C.

14.

Analyze each chromatin preparation on an agarose gel. 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 200-2000 bp.

15.

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

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Figure 4: Validation of chromatin shearing efficiency.
Chromatin preparations of HEK293T cells transfected with either pAM_dCas9 alone (no gRNA negative control) or adual transfection of the pAM_gRNA_CTCF vector with the pAM_dCas9 vector (CTCF positive control) were run on a
1.5% agarose gel. The enChIP Kit manual was used to formaldehyde fix and extract chromatin from each transfected cellline. Chromatin was then fragmented using Active Motif’s EpiShear™ Probe Sonicator and EpiShear™ Cooled Sonication
Platform to generate the desired fragmentation range of 200-2000 bp. A portion of the chromatin was used for reversalof cross-links and DNA purification. Following quantification, 500 ng of each chromatin sample was run on the agarose gel
to confirm 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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Protocols – Chromatin Immunoprecipitation
Section C. Protein G Magnetic Bead Preparation
This section describes the processing of the Protein G Magnetic beads prior to use in enChIP.
1.

In 1.5 ml microcentrifuge tubes prepare the Protein G magnetic beads. Set up separate tubes
for each IP reaction and prepare a bulk reaction for pre-clearing. If more than 4 IP reactions
will be performed, we suggest setting up multiple pre-clearing reactions to ensure sufficientroom in the tube for the addition of all the required components. Volumes to add will bebased on the number of IP reactions performed. Include negative (no gRNA) and positive
control IPs into the calculation for the number of IP reactions.
Reagent

IP Reactions

Pre-clearing Rxn

Protein G Magnetic Beads

50 µl per tube

35 µl x _____ IPs*

* We do not recommend pre-clearing more than 4 IP reactions in a single tube. If more than 4 IPs areto be performed in a single experiment, set up multiple tubes for pre-clearing.

2.

Place the tubes on a magnetic stand to pellet beads on the tube side. Wait 2 minutes andcarefully remove and discard supernatant.

3.

Remove tubes from the magnetic stand. Add 1 ml Equilibration Buffer AM2 to each tube.
Invert the tubes to mix. Quick spin to collect contents to the bottom of the tube.

4.

Place the tubes on a magnetic stand to pellet beads on the tube side. Wait 2 minutes andcarefully remove and discard supernatant.

5.

Repeat steps 3-4.

6.

Set up antibody pre-binding reactions with Protein G magnetic beads according to the tablebelow. For IP reactions, add 5 µg AM-Tag monoclonal antibody to each tube. For pre-clearing
bulk reactions, add 3.5 µg anti-mouse IgG per IP.
Reagent

IP Reactions

Pre-clearing Rxn

Blocking Buffer AM4

500 µl per tube

300 µl x _____ IPs

AM-Tag monoclonal antibody

5 µg per tube––

3.5 µg x _____ IPs

3 hours

2 hours

Anti-mouse IgG
Incubate with rotation at 4˚C

7.

Cap tubes and incubate on an end-to-end rotator at 4°C for 2-3 hours. Follow the recommendations in the chart above for the incubation time.

8.

Remove the pre-clearing reactions from the rotator and quick spin to collect contents tothe bottom of the tube. Allow the IP reactions to continue to incubate on the rotator.

9.

Place the pre-clearing tube(s) on a magnetic stand to pellet beads on the tube side. Wait 2
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minutes and carefully remove and discard supernatant
10. Remove tubes from the magnetic stand. Add 1 ml Equilibration Buffer AM2 to each tube.
Invert the tubes to mix. Quick spin to collect contents to the bottom of the tube.
11.

Place the tubes on a magnetic stand to pellet beads on the tube side. Wait 2 minutes andcarefully remove and discard supernatant.

12.

Repeat steps 10-11.

13.

Resuspend the beads with 35 µl x ______ IPs of Equilibration Buffer AM2. Pipette up anddown to completely resuspend the beads into a uniform solution. If multiple pre-clearing
reactions were used, add the Equilibration Buffer AM2 to each tube. Then, combine thepre-clearing reactions into a single tube. Pipette up and down to completely resuspend thebeads into a uniform solution.

Section D. Immunoprecipitation
1.

If working with large-scale transfections, place ChIP Buffer on ice to chill.

2.

Prepare labeled microcentrifuge tubes corresponding to each IP reaction to be performed.
Mark each tube as “pre-cleared”. Pipette bead solution up and down to ensure the beads
have not settled. Dispense 30 µl of the resuspended pre-cleared bead solution per tube.

3.

Thaw chromatin on ice. Add chromatin to its corresponding labeled pre-cleared bead tube:
For small scale transfections: Add 500 µl chromatin.
For large scale transfections: Add 200 µl chromatin and 300 µl ice-cold ChIP Buffer supplemented with 3 µl PIC and 3 µl PMSF.

4.

Return tubes to the rotator and incubate at 4°C for 1 hour.

5.

Approximately 10 minutes before the end of the pre-cleared incubation, remove the
IP reactions from the rotator and quick spin to collect contents to the bottom of the tube.

6.

Place the IP tubes on a magnetic stand to pellet beads on the tube side. Wait 2 minutes andcarefully remove and discard supernatant

7.

Remove tubes from the magnetic stand. Add 1 ml Equilibration Buffer AM2 to each tube.
Invert the tubes to mix. Quick spin to collect contents to the bottom of the tube.

8.

Place the tubes on a magnetic stand to pellet beads on the tube side. Wait 2 minutes andcarefully remove and discard supernatant.

9.

Repeat steps 6-7.

10. Place washed IP beads on ice.
11.

Following the 1 hour incubation of the pre-cleared beads with the chromatin, remove thetubes from the rotator and quick spin to collect contents to the bottom of the tube.

12.

Place the pre-cleared reactions on a magnetic stand for 2 minutes to pellet beads on the
tube side.
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13.

Carefully transfer the supernatant from the pre-cleared beads with the chromatin to thecorresponding IP reactions containing the AM-tag antibody-conjugated beads on ice.

14.

Return the reactions to an end-to-end rotator and incubate overnight at 4°C.

Section E. Washing of IP reactions
We recommend using chilled buffers for the IP wash steps. Place buffers on ice prior to use.
1.

Remove the IP reactions from the rotator and quick spin to collect contents to the bottomof the tube.

2.

Place the tubes on a magnetic stand to pellet beads on the tube side. Wait 2 minutes andcarefully remove and discard supernatant.

3.

Remove tubes from the magnetic stand. Add 1 ml ice-cold LS Buffer to each tube and invert
to mix.

4.

Incubate on an end-to-end rotator at 4°C for 10 minutes. Remove tubes from the rotator andquick spin to collect contents to the bottom of the tube.

5.

Place the tubes on a magnetic stand to pellet beads on the tube side. Wait 2 minutes andcarefully remove and discard supernatant.

6.

Repeat steps 3-5.

7.

Remove tubes from the magnetic stand. Add 1 ml ice-cold Wash Buffer AM1 to each tube
and invert to mix.

8.

Incubate on an end-to-end rotator at 4°C for 10 minutes. Remove tubes from the rotator andquick spin to collect contents to the bottom of the tube.

9.

Place the tubes on a magnetic stand to pellet beads on the tube side. Wait 2 minutes andcarefully remove and discard supernatant.

10. Repeat steps 7-9.
11.

Remove tubes from the magnetic stand. Add 1 ml ice-cold LiCl Buffer to each tube andinvert to mix.

12.

Incubate on an end-to-end rotator at 4°C for 10 minutes. Remove tubes from the rotator andquick spin to collect contents to the bottom of the tube.

13.

Place the tubes on a magnetic stand to pellet beads on the tube side. Wait 2 minutes andcarefully remove and discard supernatant.

14.

Repeat steps 11-13.

15.

Remove tubes from the magnetic stand. Add 1 ml ice-cold TE to each tube and invert to mix.

16.

Incubate on an end-to-end rotator at 4°C for 10 minutes. Remove tubes from the rotator andquick spin to collect contents to the bottom of the tube.

17.

Place the tubes on a magnetic stand to pellet beads on the tube side. Wait 2 minutes andcarefully remove and discard supernatant.
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2118. Repeat steps 15-17.

Section F. Elution, Reversal of Cross-links and DNA Purification
1.

Remove tubes from the magnetic stand. Resuspend each IP in 100 µl Elution Buffer AM4.
Pipette up and down to mix.

2.

Briefly vortex samples and incubate at room temperature for 15 minutes without rotation.

3.

Briefly vortex samples and quick spin to collect contents to the bottom of the tube. Placethe tubes on a magnetic stand for 2 minutes to pellet beads on the tube side.

4.

Transfer supernatant to 0.2 ml PCR tubes.

5.

Add 2 µl Proteinase K. Vortex to mix. Heat samples in a thermocycler at 55°C for 30 minutes.
Then increase the temperature to 80°C for 2 hours.

6.

Transfer the DNA to a 1.5 ml microcentrifuge tube and add 5 volumes (500 µl) DNA Purification Binding Buffer to each tube. Vortex to mix. Adjust the pH with 5 µl 3 M Sodium Acetate.
The sample should be bright yellow in color to indicate proper pH. If your sample is notbright yellow, please refer to the Troubleshooting guide in the Appendix on page 26 fordetails to adjust pH prior to loading the sample into the purification column.

7.

For each sample, place a DNA purification column (AM# 103928) in a collection tube and add
each pH adjusted sample to its own column. Close the cap on each column and centrifuge at
14,000 rpm for 1 minute.

8.

Remove the column from the collection tube, then remove and discard the flow throughfrom the collection tube. Return the column to the collection tube.

9.

Prepare DNA Purification Wash Buffer (AM #103497) before the first use. Follow the instruction on page 14 for the addition of ethanol prior to using the solution. Add 750 µl DNA
Purification Wash Buffer to each column and cap the column.

10. Spin at 14,000 rpm for 1 minute in a microcentrifuge.
11.

Pre-warm DNA Purification Elution Buffer (36 µl x _____IPs) at 37°C for 5 minutes prior to
use.

12.

Remove the column from the collection tube, then remove and discard the flow throughfrom the collection tube. Return the column to the collection tube.

13.

With the column cap open, spin at 14,000 rpm for 2 minutes in a microcentrifuge to remove
any residual Wash Buffer from the column.

14.

Transfer the column to a clean microcentrifuge tube. Add 36 µl pre-warmed elution buffer
directly to the center of each column. Let sit for 1 minute.

15.

Spin at 14,000 rpm for 1 minute in a microcentrifuge.

16.

Discard column. Purified DNA may be stored at -20°C for future use. We recommend preparing a DNA dilution for use in qPCR (Add 6 µl eluted DNA to 94 µl sterile water). The remaining 30 µl DNA sample can be used for library generation and sequencing.
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Protocols – ChIP DNA Analysis
Section G: qPCR Primer Design
Guidelines are provided below to design primers for use in qPCR analysis of the enriched DNA.
• 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.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 (Catalog No. 53029), primers should be designed
to anneal optimally at 58°C with a recommended length of 18-22 bp.

Section H. Quantitative PCR (qPCR)
Active Motif recommends the use of its ChIP-IT® qPCR Analysis Kit (Catalog No. 53029) for the
analysis of qPCR data. The ChIP-IT qPCR Analysis Kit contains positive and negative control primer
pairs, standard curve DNA, standard curve primers and a qPCR Analysis spreadsheet to performthe analysis calculations. Active Motif’s analysis strategy determines primer efficiencies and theChIP sample values are normalized according to input, primer efficiency, chromatin amount usedin the ChIP reaction and resuspension volume. The ChIP-IT qPCR Analysis Kit provides consistency
in data analysis and allows direct comparison across samples and experiments.
If using the ChIP-IT qPCR Analysis Kit, please refer to the product manual for instructions.
If the data analysis will not be performed using Active Motif’s ChIP-IT qPCR Analysis Kit, prepare astandard curve using known amount of Input DNA for each primer pair being used in the experiment. Calculate the fold enrichment of positive primers (known binding sites) relative to negative
control primers (regions of the genome not bound by your gRNA of interest).
1.

Produce a standard curve by performing qPCR with your primer set on known DNA
quantities of Input DNA (from Section B) in triplicate. Run three to five samples that are
10-fold dilutions, e.g. 0.005 ng, 0.05 ng, 0.5 ng, 5 ng and 50 ng.

2.

Run the enChIP and no gRNA samples along with the dilution series of the Input DNA
standards using both positive control primers and negative control primers.
Input DNA: Prepare dilution series based on input DNA concentration. Use 5 µl per
PCR reaction.
enChIP / no gRNA DNA: Dilute 6 µl of the 36 µl elution volume into 94 µl sterile water.
Use 5 µl of diluted sample per PCR reaction.

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3.

Below is an example qPCR reaction. Please follow the specific instructions for yourqPCR instrument. We recommend using a commercially available SYBR Green qPCR
master mix (e.g. Bio-Rad Cat # 170-8882) and preparing triplicate reactions.
Reagent4.
		

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 (enChIP or Input)

5 µl

Total volume

20 µl

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

5.

Your qPCR instrument will assign values (in ng) to each qPCR reaction based on thestandard curve. If your machine does not average your triplicate reactions automatically, you will need to calculate these averages.

6.

Divide the average value from the positive control primer set by the average value ofthe negative control primer set to obtain your fold enrichment.

Figure 5: qPCR analysis of positive control CTCF binding site using the enChIP Kit.
The positive control pAM_gRNA_CTCF Vector (Catalog No. 53123), which contains a gRNA sequence corresponding to a
CTCF binding site on chromosome 19 (5,804,115–5,804,209), and the pAM_dCas9 (no gRNA) negative control vector weretransfected into HEK293T cells. Chromatin was prepared and immunoprecipitated using the AM-tag antibody according to
the instructions in the enChIP Kit. Enriched DNA was analyzed by qPCR to evaluate a negative control (Sox2 promoter) andpositive control (CTCF) gene locus. Results show enrichment of CTCF at the expected gene locus and little to no enrichment of the no gRNA negative control or Sox2 promoter.

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24Section I. ChIP-Seq
ChIP-seq analysis involves the preparation of libraries from ChIP DNA by the addition of adapter
sequences to the ends of the DNA fragments. The library is then PCR amplified and validated prior
to sequencing. For enChIP, we recommend using the Illumina® platform. Create TruSeq libraries
and perform sequencing using Illumina NextSeq or HiSeq. We recommend sequencing 50-75 bp
single end reads for a total of at least 20 million reads. The no gRNA samples should be sequenced
as a control reaction in order to identify false “peaks” associated with the dCas9 protein and also
to reveal regions of the genome that have been duplicated. Subtracting the no gRNA peaks fromthe experimental peaks will help eliminate false data.

CTCF gRNA1

CTCF gRNA2

No gRNA

Total number of reads

39,114,618

40,036,875

38,208,604

Total number of alignments

31,486,919

29,584,781

31,765,380

Unique alignments

28,729,643

26,987,217

28,634,570

Unique alignments (without duplicate reads)

27,418,487

24,458,100

26,837,038

100

30,822

N/A

Peak numbers

Figure 6: ChIP-Seq analysis of CTCF binding site using the enChIP Kit.
Two gRNAs were designed using CRISPRdirect targeting different sequences of a CTCF binding site on chromosome 19. EachgRNA sequence was cloned into the pAM_dCas9 Vector and transfected into HEK293T cells along with a no gRNA negative
control transfection. Chromatin was prepared and immunoprecipitated according to the instructions in the enChIP Kit.
Enriched DNA was analyzed by ChIP-seq and background was subtracted using the no gRNA control. Data was compared
to traditional ChIP-seq data for CTCF in the same cell line. Results show gRNA 1 had a strong peak at the desired target siteand had little to no off-target binding events. Data for gRNA 2 revealed a larger number of peaks, which represented nonspecific binding of the gRNA outside of the target locus. A comparison of overlapping peak data for gRNA 1, gRNA 2 andthe ChIP-seq data set could provide information about potential looping interactions.

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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 dounce homogenization and centrifugation (intact nuclear pellet), -80°C.
3. After chromatin shearing, -80°C.
4. After DNA clean up, -20°C.

Poor yield of shearedchromatin.

Insufficient cell numbers were used. Repeat chromatin preparation using a larger numberof 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.
Buffers were not scaled proportionally to the size of the sample. Use the recommendations for cell culture size.

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.
Chromatin should be between 200-2,000 bp in size. If you observe higher molecularweight products, we recommend to decrease the size of the fragments by re-sonicating
the sample. If chromatin falls within the lower end of the size range (e.g. 200-700 bp) thisis still sufficient for determining gRNA specificity. However, these shorter fragments may
not provide reliable information regarding chromatin looping.

Performing enChIP with alarge volume of chromatin.

This is not recommended. Follow the instructions in the manual for chromatin quantities
based on the cell culture volumes. Do not perform a single scaled-up reaction, as thecapture efficiency is lower.

enChIP DNA does not turnbright yellow following theaddition of 3 M sodium
acetateIf the color is light orange or violet, this indicates the pH is too high. Add more 3 M
sodium acetate 5 µl at a time, mixing after addition until the color is bright yellow. This
step is crucial to the success of DNA binding and purification. For a full color image please
see the manual for Active Motif’s Chromatin IP DNA Purification Kit Catalog No. 58002
available online at our website www.activemotif.com.

High background.

Chromatin not sheared enough. Shearing should produce DNA fragments that are smallenough to exclude background from neighboring chromosomal sequences, but still large
enough that there is a good possibility your amplicon remains intact. We recommend
200-2,000 bp fragments. If the DNA fragments are too large, the background is increased.
Consider increasing the number of pulses for sonication. Check the fragment size on a gel
to assess your shearing efficiency.
Non-specific gRNA design. Some gRNAs display off-target binding, resulting in highbackground. It is recommended to design multiple gRNAs to a specific genomic locus andtest them in enChIP to identify the most specific gRNA. Use a software program to design
a new gRNA target sequence.

www.activemotif.com

26

Problem/question

Recommendation

Poor or no enrichmentwith AM-Tag monoclonal
antibody.

Ensure that you are using the correct antibody. The AM-Tag antibody provided in the kit
is a mouse monoclonal antibody. The antibody recommended for WB analysis of protein
expression (Catalog No. 61677) is a rabbit polyclonal antibody. The rabbit polyclonal
antibody is not validated for use in enChIP. Use the AM-Tag monoclonal antibody.
Inefficient transfection efficiency or poor expression of the gRNA and dCas9 vectors.
Prepare a duplicate transfection reaction. Prepare cell lysates and analyze for expressionof the AM-tagged dCas9 protein by Western blot using Active Motif’s AM-Tag polyclonal
antibody (Catalog No. 61677). If the tagged dCas9 protein is not detected, continue to
optimize transfection conditions.
Problems with PCR. Confirm the amplified sequence for the positive control primer set
contains the genomic region targeted by the gRNA. Design new PCR primers.
Run a positive control reaction using Active Motif’s pAM_gRNA_CTCF vector (Catalog
No. 53123) or the pAM_dCas9_CTCF vector (Catalog No. 53124) to confirm all other assay
reagents are working.

No PCR products withreal-time PCR

Confirm the species specificity and efficiency of your primers. You may need to redesign
your primers. Primers that work in end point PCR do not always work in qPCR.
Ensure that your PCR amplicon is designed to include your gRNA target sequence. Referto your gRNA design. Make sure your gRNA target sequence does not contain any known
binding sites of DNA binding proteins. Protein binding may interfere with the ability ofyour gRNA to bind its genomic locus.
No ethanol in DNA Purification Wash Buffer. Make sure that ethanol has been added to
the DNA Purification Wash Buffer prior to first use.

www.activemotif.com

27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		
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
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
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		
Modified Histones Array		50 rxns
25 rxns
Format

54001
54002
Catalog No.

MODified™ Histone Peptide Array		

1 array

13001

Histone Modification FP Binding Assay		

FormatCatalog No.

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

1 kit

57001

www.activemotif.com

28

Histone ELISAs		FormatCatalog No.

Histone H3 monomethyl Lys4 ELISA		Histone H3 dimethyl Lys4 ELISA		Histone H3 trimethyl Lys4 ELISA		Histone H3 acetyl Lys9 ELISA		Histone H3 dimethyl Lys9 ELISA		Histone H3 trimethyl Lys9 ELISA		Histone H3 phospho Ser10 ELISA		Histone H3 acetyl Lys14 ELISA		Histone H3 monomethyl Lys27 ELISA		Histone H3 trimethyl Lys27 ELISA		Histone H3 phospho Ser28 ELISA		

1 x 96 rxns
1 x 96 rxns
1 x 96 rxns
1 x 96 rxns
1 x 96 rxns
1 x 96 rxns
1 x 96 rxns
1 x 96 rxns
1 x 96 rxns
1 x 96 rxns
1 x 96 rxns

53101
53112
53113
53114
53108
53109
53111
53115
53104
53106
53100

Histone Purification & Chromatin Assembly		

FormatCatalog No.

Histone Purification Kit		
Histone Purification Mini Kit		
Chromatin Assembly Kit		
HeLa Core Histones		

10 rxns
10 rxns
10 rxns
36 µg

40025
40026
53500
53501

Recombinant Methylated, Acetylated and Phosphorylated Histone Proteins
For an up-to-date list of Recombinant Histone Proteins, please visit www.activemotif.com/recombhis.
Histone Acetyltransferase and Deacetylase Activity

FormatCatalog No.

HAT Assay Kit (Fluorescent)		
Recombinant p300 protein, catalytic domain		
Recombinant GCN5 protein, active		
HDAC Assay Kit (Fluorescent)		
HDAC Assay Kit (Colorimetric)		

1 x 96 rxns
5 µg
5 µg
1 x 96 rxns
1 x 96 rxns

56100
31205
31204
56200
56210

Histone Demethylase Activity		FormatCatalog No.

Histone Demethylase Assay (Fluorescent)		

48 rxns

53200

DNA Methylation		

FormatCatalog No.

hMeDIP		
MeDIP		MethylDetector™		
MethylCollector™		
MethylCollector™ Ultra		
UnMethylCollector™		
Hydroxymethyl Collector™		
DNMT Activity / Inhibition Assay		
Methylated DNA Standard Kit		
Fully Methylated Jurkat DNA		
Jurkat genomic DNA		

10 rxns
10 rxns
50 rxns
25 rxns
30 rxns
30 rxns
25 rxns
96 rxns3 x 2.5 µg
10 µg
10 µg

55010
55009
55001
55002
55005
55004
55013
55006
55008
55003
55007

www.activemotif.com

29

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
Azuma Bldg, 7th Floor
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

Visit Active Motif on the worldwide web at http://www.activemotif.com
At this site:• Read about who we are, where we are, and what we do• Review data supporting our products and the latest updates• Enter your name into our mailing list to receive our catalog, MotifVations newsletter andnotification of our upcoming products• Share your ideas and results with us• View our job opportunities
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