NOMe-Seq

Catalog No. 54000(version A2)

Active Motif North America
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Copyright 2012 Active Motif, Inc.

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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.
This documentation may not be copied, transferred, reproduced, disclosed, or duplicated, inwhole or in part, without the prior written consent of Active Motif, Inc. This documentation is
proprietary information and protected by the copyright laws of the United States and international treaties.
The manufacturer of this documentation is Active Motif, Inc.
© 2012 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
Flow Chart of Process  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  . 2Introduction  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Traditional Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Kit Performance and Timeline .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  . 5Kit Components and Storage .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  . 6Additional Materials Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Protocols – Experimental Set Up
Notes Before Starting  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Bisulfite-Specific PCR Primer Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Buffer Preparation and Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Protocols – NOMe-Seq
Section A. Cell Fixation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Section B. Preparation of Sheared Chromatin by Sonication . . . . . . . . . . . . . . . . . . . . . . . .  14
Section C. Methyltransferase Reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Section D. Proteinase K Treatment and DNA Purification . . . . . . . . . . . . . . . . . . . . . . . . . . .  16
Section E. Bisulfite Conversion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Section F. On-Column Desulfonation and DNA Purification . . . . . . . . . . . . . . . . . . . . . . . .  18
Section G. PCR Reactions to Amplify Genomic Region of Interest . . . . . . . . . . . . . . . . . . .  18
Section H. Purification of Amplified DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  19
Section I. DNA Cloning and Sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Section J. Data Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Appendix
Section K. Troubleshooting Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
Section L. Related Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Technical Services  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

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Overview
Understanding the relationship between nucleosome occupancy, transcription factor binding and
DNA methylation profiles is a complex system that is not well understood. Epigenome mapping
studies have shown nucleosome depleted regions at transcription start sites and enhancers, but
their role in gene regulation is not clear. To better interrogate the function of nucleosome occupancy in the context of DNA methylation, Active Motif offers a high resolution single-molecule
Nucleosome Occupancy and Methylome sequencing (NOMe-Seq) Kit. The NOMe-Seq assay allows
for simultaneous analysis of nucleosome occupancy and CpG methylation on the same DNA
strand1,2,3.
The NOMe-Seq assay works by treating fixed chromatin with a GpC DNA methyltransferase
enzyme to artificially methylate GpC dinucleotides that are not protected by nucleosomes orprotein-DNA binding. Following DNA extraction and bisulfite conversion, genomic regions ofinterest are PCR amplified, cloned and sequenced. Alternatively, for larger genome analysis, NextGen libraries can be generated and sequenced4. The readout of the GpC methylation provides afootprint of nucleosome occupancy in combination with the endogenous CpG DNA methylation
profile for the same DNA strand. By utilizing fixed chromatin in which all protein-DNA binding
interactions are preserved, NOMe-Seq also offers the added benefit of providing preliminary
information regarding transcription factor binding sites.
Enough reagents are provided in the NOMe-Seq Kit to perform 10 enzymatic treatments and 10
negative control reactions. Each GpC Methyltransferase treatment is performed on chromatinfrom 250,000 cell equivalents.

product

formatcatalog no.

NOMe-Seq

10 rxns*

54000* Licensed technology. Patent pending.

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1Flow Chart of Process

NOMe-Seq Method
Fix cells & shear chromatin by sonication to > 1 kb
CpG sites
GpC sitesCpG sites

Treat with GpC Methyltransferase
CpG sites
GpC sitesCpG sites

Reverse cross-links/DNA purification
Bisulfite conversion
PCR amplification/cloning, followed by Sequencing
CpG sites
GpC sites≥ 147 bp
Nucleosome

10-80 bp
ProteinUnmethylated≥ 147 bp
Nucleosome

Methylated≥ 147 bp
Nucleosome

Nucleosome

Bound transcription factor

Flow Chart of NOMe-Seq Methodology
In NOMe-Seq, cells are formaldehyde fixed to cross-link protein/DNA interactions and preserve the location of the nucleosomes. The chromatin is then sonicated to generate fragments of 1 kb or greater for analysis. Treatment of chromatinwith a GpC Methyltransferase enables artificial methylation of GpC dinucleotides that are not protected by nucleosomes
or DNA-bound proteins. Following enzymatic treatment, the chromatin is subjected to a reversal of cross-links, treatment
by Proteinase K, RNase A and DNA purification. Bisulfite conversion is then performed on the DNA to provide single-nucleotide resolution of the methylation profile. For locus-specific analysis, bisulfite converted PCR primers are used to PCR
amplify the region of interest which is cloned and sequenced. Alternatively, Next-Gen sequencing methods can be used.
By plotting the GpC regions of the locus of interest and analyzing the methylation profile, areas in which GpC methylation
occurred (black circles) will represent open chromatin regions that were accessible to enzymatic treatment. GpC sites thatwere not methylated (white circles) represent regions of the chromatin that were protected from enzymatic treatment by
the presence of nucleosomes or protein. To overlap the chromatin state with the DNA methylation profile for the samegene, the endogenous CpG methylation from the bisulfite sequencing data can be analyzed. The NOMe-Seq method
therefore allows for information to be simultaneously determined regarding nucleosome occupancy and CpG methylation
for the same DNA strand.

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Introduction
Nucleosome occupancy and gene regulation
The genomes of eukaryotic organisms are packaged into chromatin. The nucleosome is thesmallest subunit of chromatin and consists of 147 base pairs of DNA wrapped around an octamerof core histone proteins (two each of Histone H2A, Histone H2B, Histone H3 and Histone H4).
Histone H1 exists as a linker histone present at the interface between the nucleosome core andDNA entry/exit points.
Chromatin is subject to a variety of chemical modifications, including post-translational modifications of the histone proteins and the methylation of cytosine residues in the DNA. These
modifications play a major role in regulating gene expression by modifying the accessibility ofpromoter regions to transcriptional machinery5,6. Transcriptional co-activator complexes interact
with nucleosomes, and these interactions induce nucleosomal rearrangements to unfold the nucleosome completely or disassemble it at the transcriptional start site (TSS) to allow for transcription initiation7,8,9,10. The presence of the nucleosome depleted region (NDR) upstream of the TSS is
critical for gene activation11.
DNA methylation in mammalian cells is generally associated with gene silencing, either directly
by inhibiting binding of transcription factors to their recognition sequences12, or indirectly by
preventing transcription factors from accessing their target sites through attachment of methylCpG binding proteins (MBD) that recruit histone deacetylases and histone methyltransferases.
Mechanisms such as ATP-dependent chromatin remodeling work to alter chromatin structure inorder to regulate DNA accessibility13.
Classical techniques used to understand nucleosome positions, such as nuclease digestion andprimer extension14, often destroy the physical linkages between binding sites and prevent direct
analysis of single molecule promoter function. Instead these technologies look at the average
distribution across a panel of remodeled nucleosomes15. Active Motif’s NOMe-Seq Kit provides amethod to obtain single molecule analysis of nucleosome positioning.
In NOMe-Seq, a GpC methyltransferase enzyme is used to obtain nucleosome positioning
information. The enzyme artificially methylates GpC residues not protected by nucleosomes ortranscription factor binding. Due to the abundance of GpC dinculeotides in the genome, their
methylation profile enables high resolution of nucleosome position. Additionally, GpCs are not endogenously methylated in mammalian genomes, so artificial methylation does not interfere withthe ability to establish an endogenous methylation profile. Following enzymatic treatment, thechromatin is subjected to a reversal of cross-links, treatment by RNaseA, Proteinase K and DNA
purification. The purified DNA undergoes bisulfite conversion to obtain the methylation profile
of each DNA strand. The NOMe-Seq reactions are then PCR amplified to analyze the region ofinterest, cloned and sequenced. The sequencing results provide information on both nucleosome
position and CpG methylation to reveal the relationship between multiple epigenetic modifications on the same DNA molecule. A comparison to negative control reactions will resolve if themethylation status of GpCpG trinucleotides is due to artificial or endogenous methylation.
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Traditional Methods to Study Nucleosome Positioning or DNA
Methylation
To date, there are several methods used to study nucleosome positioning or DNA methylation
independently:
1. Nuclease Digestion: The use of enzymes, such as micrococcal nuclease (MNase) or DNaseI,
to digest DNA followed by sequencing or PCR, is often used to map the position of nucleosomes. The technique is based on the fact that protein bound to DNA will often protect
the DNA from enzymatic cleavage. Therefore, enzymes will digest linker DNA connecting
two nucleosomes and the resulting digestion pattern reveals nucleosome positioning. However, these methods require significant amounts of material, are variable based on enzyme
concentration and can introduce bias into the system based on cutting preferences.
2. FAIRE: Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) is a method used toenrich for nucleosome depleted DNA using formaldehyde fixation and phenol-chloroform
extraction16. This method relies on the idea that formaldehyde cross-linking is more efficientin nucleosome-bound DNA, therefore DNA cross-linked to nucleosomes is sequestered in
the interphase, whereas DNA recovered in the aqueous phase corresponds to nucleosomedepleted regions of the genome. The nucleosome-free DNA can be sequenced to identify
regions that correspond with transcriptional start sites, enhancers and active promoters.
3. Bisulfite conversion: Bisulfite conversion consists of the treatment of genomic DNA withsodium bisulfite, leading to deamination of unmethylated cytosines into uracil. PCR is thenperformed with primers that differentiate between methylated and unmethylated sequences (Methylation-specific PCR (MSP)), or the bisulfite converted DNA is sequenced to reveal
the methylation profile at single-nucleotide resolution. One draw back of this technique isthat bisulfite conversion cannot distinguish between 5-methylcytosine and 5-hydroxymethylcytosine.
4. MAP: Methyltransferase Accessibility Protocol (MAP) utilizes DNA methyltransferases as ameans to map nucleosomes and non-histone protein-DNA interactions17. The accessibilityof the DNA for the methyltransferase is obtained through thermal cycle sequencing with aprimer specific to the strand of interest18. The result is the relative frequency of accessibility across a population. The MAP technology has also been expanded to MAP for individual
templates (MAPit), which is a single-molecule approach to determine the CpG methylationof mammalian genes and their overlaying chromatin structure18, 19. MAP-IT uses a combinationof a cytosine DNA methyltransferase enzyme and bisulfite sequencing to map protein-DNA
interactions.

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4Kit Performance and Timeline
NOMe-Seq is intended for locus-specific analysis of nucleosome positioning in combination with
the DNA methylation profile of the locus of interest. PCR is used to amplify the region of interest
for Sanger sequencing analysis. However, if a larger portion of the genome is to be analyzed, theNOMe-Seq assay can be combined with Next-Gen sequencing using the adapter ligation protocol
provided at www.activemotif.com/nome-seq.
Day 1

Cell fixation with formaldehyde
Cell lysis and nuclei collection
Prepare 750,000 cell equivalents for sonication
Sonicate chromatin into fragments greater than 1 kb
Treat 250,000 cell equivalents with GpC methyltransferase for 4 hrs at 37˚COvernight reversal of cross-links

Day 2Treat with RNaseA and Proteinase K
Purify genomic DNA
Bisulfite conversion of DNA for 5 hrs at 50˚C

Day 3Desulfonation and DNA purification of bisulfite treated DNA
PCR amplification of genomic region of interest with bisulfite-specific PCR primers
Purify PCR amplicon of interest

Day 4 +
Ligation of PCR product into cloning vector

Perform end repair

Transformation of ligated DNA vector

Concatemerization of amplicons

Selection of colonies for screening

Sonication of concatemerized sample

PCR amplify to screen for correct insert

Perform end repair

Isolation of plasmid DNA from correct clones

Add A overhang to 3’ ends

Submit DNA samples for Sanger sequencing

Ligate adapters
Size select library
Enrich the adapter-modified DNA
Validate the library
Submit DNA samples for Next-Gen sequencing

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NOMe-Seq Kit Components and Storage
NOMe-Seq Kits are for research use only. Not for use in diagnostic procedures. Kits are shipped in
two boxes and contain reagents with multiple storage temperatures inside. Please store each component at the temperature listed below. The product is stable for 6 months when stored properly.
Reagents

Quantity

Storage

10X PBS

100 ml4°C

10X Glycine

40 ml

-20°C

100 mM PMSF

500 µl-20°C

Protease Inhibitor Cocktail (PIC)

100 µl

-20°C

Lysis Buffer

20 ml

-20°C

10X NE Wash Buffer

5 ml

4°C

10X Reaction Buffer AM2

500 µl4°C

1 M DTT

100 µl-20°C

100X AdoMet

120 µl-20°C

GpC Methyltransferase enzyme (4 U/µl)

50 µl

-20°C

Stop Solution AM2

1 ml

RT

RNase A (10 mg/ml)

2 x 40 µl-20°C

Proteinase K (10 mg/ml)

200 µl-20°C

DNA Purification Binding Buffer (AM #103496)

50 ml

RT

DNA Purification Wash Buffer (AM # 103497)*

50 ml

RT

DNA Purification Elution Buffer (AM #103498)

5 ml

RT

3 M Sodium Acetate**

500 µlRT

Genomic DNA purification columns (AM # 103945)

20 ea

RT

Conversion Reagent

5 ea

RT

Buffer A

2 ml

RT

Denaturation Reagent

1 ml

RT

Hydroquinone

5 ea

RT

Buffer B

1 ml

4°C

DNA Purification Columns (AM #101037)

50 ea

RT

DNA Binding Buffer (AM #101232)

15 ml

RT

DNA Wash Buffer (AM #101233)

25 ml

RT

DNA Elution Buffer (AM #100762)

3 ml

RT

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* DNA Purification Wash Buffer must be reconstituted to a final concentration of 80% ethanol
prior to use.
** Do not store the 3M Sodium Acetate at 4°C.
Additional materials required but not supplied• Cell culture reagents for growing cell lines• 37% Formaldehyde• Microprobe tip sonicator (Active Motif’s Epishear Sonicator, Catalog No. 53051 and EpiShear
Cooled Sonication Platform, Catalog No. 53073)• Hemocytometer & Trypan Blue for cell counting• Cell scraper (rubber policeman)• Dounce homogenizer (Active Motif, Catalog No. 40401)• Incubators for 37°C, 50°C, 65°C and 95°C temperatures• Ethanol, 100%
• Isopropanol, 100%
• 1.5 and 2 ml microcentrifuge tubes and microcentrifuge• Thermocycler• 0.2 ml PCR tubes and caps
• PCR reagents (Bioline 2X MyTaq™ Master Mix, Catalog # BIO-25041, or individual PCR reagents
such as dNTPs, PCR Buffer and Taq polymerase)• Gel extraction kit (Optional)• Phenol/Chloroform/Isoamyl Alcohol (Optional)• DNA vector for PCR fragment cloning and ligation reagents• Competent E. coli cells for DNA cloning (Active Motif’s RapidTrans™ TAM1 chemicallycompetent cells, Catalog No. 11096)• DNase-free sterile water
Notes:The polymerase chain reaction (PCR) process for amplifying nucleic acid is covered by U.S. Patent Nos. 4,683,195 and 4,683,202 assigned to Hoffmann-La Roche.
Patents pending in other countries.
Use of methylation-specific PCR (MSP) is protected by U.S. Patent Nos. 5,786,146,
6,017,704, 6,200,756 & 6,265,171 and International patent WO97/46705. No licenseunder these patents to use the MSP process is conveyed to the purchaser bypurchasing this product.

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Protocols – Experimental Set Up
Notes Before Starting
It is important to read through the entire manual before starting the protocol. The NOMe-Seq
Assay is designed to map the location of nucleosomes to the chromatin structure. A comparisonof samples treated with the GpC methyltransferase enzyme and those not treated will enable theanalysis of endogenous methylation in addition to nucleosome positioning. The assay includes
enough reagents to perform 10 enzymatic treatments and 10 negative control reactions. The assay
is intended for locus-specific analysis.
To study the nucleosome positioning pattern of a specific region, it is necessary to design PCRprimer mixes for bisulfite converted DNA. We recommend that primer design is performed prior
to starting NOMe-Seq. Recommendations on bisulfite-specific primer design are listed below.
The approximate time frame to complete the NOMe-Seq Assay including chromatin preparation,
enzymatic treatment of chromatin, bisulfite conversion of genomic DNA, PCR amplification of
the gene locus of interest and DNA cloning is 5 days. The protocol does provide stopping points
throughout the assay.
Please check the list of additional materials required but not provided prior to starting the protocol. Suggested protocols are provided for PCR amplification and DNA cloning, but we recommend
you follow the manufacturer’s instructions for your specific PCR and cloning reagents.

Bisulfite-Specific PCR Primer Design
We recommend designing PCR primers specific for bisulfite converted DNA using an in silico
program such as the free program Methylviewer, http://dna.leeds.ac.uk/methylviewer/. MethylViewer provides an integrated solution for primer design and analysis of bisulfite sequencing18.The
GpC methyltransferase treatment that is performed on the chromatin sample prior to bisulfite
conversion will methylate all the accessible cytosines in a GpC context. These cytosines will remain as cytosine following the bisulfite treatment. Additionally, any endogenous DNA methylation
in a CpG context will also remain as cytosine following the bisulfite treatment.
The region of interest for analysis should contain a high GpC density in order to increase the resolution of the nucleosome footprint. Forward and reverse primer design should flank the GpC andCpG region of interest, but avoid containing GpC and CpG within the primer sequence itself.
The optimal amplicon length for Sanger sequencing analysis should be between 400-600 bp. This
length will allow for enough resolution to identify regions of nucleosome or transcription factor
occupancy while still enabling complete reads by single-pass sequencing.
Once bisulfite-specific PCR primers are designed, they should be tested to confirm their specificity for converted DNA. To do this, use non-converted genomic DNA as a PCR template withthe primer pair to ensure there is no DNA amplification. The primers should only produce a PCR
amplicon if the DNA has been bisulfite treated.
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8Buffer Preparation and Recommendations
Samples
The assay is intended for locus-specific analysis. The assay has been optimized for use on cultured
cell lines, but has not been tested on tissue samples.
Our standard chromatin preparation protocols use cells grown in one 15 cm tissue culture plate(approximately 1.5 x 107 cells) and yield enough material to perform up to 20 reactions. Dependingon your experiments, you may wish to work with different volumes of cells. The comments andtable below are designed to help adapt our protocols for using different cell numbers.
• It is not recommended to use a sample with less than 500 µl or more than 2 ml of the
Lysis Buffer for the cell lysis and dounce homogenization steps.
• If you intend to compare results from various samples, treat the samples equally. For
example, grow induced and uninduced cells in the same size plate and to the same density,then use equal volumes and shearing conditions. This will help ensure that the chromatin
preparations are equivalent.
• Each human diploid cell contains 6.6 picograms of DNA. This can be used to estimate DNA ina chromatin preparation if the number of cells in the starting material is known. We estimate
DNA recovery of chromatin shearing to be about 60-70%, depending on the cell type.
• Chromatin sonication is performed on 750,000 cell equivalents in a final volume of 300 µl.
If using plate sizes smaller than 15 cm plate, it may be necessary to pool multiple plates
together to obtain enough chromatin material.
• Due to the sensitivity and stability of some of the bisulfite conversion reagents, we recommend performing the NOMe-Seq Assay with a minimum of 4 samples (2 GpG enzymaticallytreated and 2 negative control reactions) per experiment. This experimental design can beused for a total of 5 experiments, or 10 enzymatically treated reactions.
1 well of a
6-well plate

60 mm dish100 mm dish15 cm plate

Number of Cells

0.9 x 106

2.4 x 106

6.6 x 106

15.0x 106

Fixation Solution

3 ml

5 ml

10 ml

20 ml

Glycine Stop-Fix

1.5 ml

2.5 ml

5 ml

10 ml

1X PBS2 x 3 ml2 x 5 ml2 x 5 ml2 x 10 ml

Cell Scraping
Solution + PMSF

500 µl +2.5 µl PMSF

1 ml +
5 µl PMSF

1 ml +
5 µl PMSF

5 ml +
30 µl PMSF

Lysis Buffer +PIC + PMSF

500 µl + 2.5 µl PIC +2.5 µl PMSF

500 µl + 2.5 µl PIC +2.5 µl PMSF

500 µl + 2.5 µl PIC +2.5 µl PMSF

1 ml + 5 µl PIC +5 µl PMSF

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Recommendations are offered in the manual for an approximate time frame of the assay. Below
are the buffer preparations required for each day of the protocol. Adjust buffer preparation asneeded based on your specific experiment.

Day 1 Buffer Preparation
Fixation Solution
Add 0.54 ml 37% formaldehyde (not included in the kit) to 20 ml minimal cell culture medium andmix thoroughly. Leave at room temperature.
1X PBS Solution
Dilute 10X PBS in dH2O. Add 2.33 ml 10X PBS to 21 ml sterile water, mix and place on ice.
Glycine Stop-Fix Solution
Combine 1 ml 10X Glycine Buffer, 1 ml 10X PBS and 8 ml sterile water. Mix well and leave at room
temperature.
Cell Scraping Solution
Add 0.6 ml 10X PBS to 5.4 ml dH2O, mix and place on ice.
Lysis Buffer
Is provided ready to use.
1X NE Wash Buffer
Prepare a 1X solution by diluting 10X NE Wash Buffer 1:10 in dH2O. For example, add 250 µl 10X NE
Wash Buffer to 2.25 ml dH2O. Mix thoroughly.
100 mM DTT
Prepare a fresh 100 mM working stock of DTT from the provided 1 M DTT each time reactions
are set up. For every 750,000 cell pellet, dilute 10 µl 1M DTT into 90 µl dH2O. Vortex to mix andplace on ice. Avoid multiple freeze thaws of DTT as this will diminish its effectiveness. Discard any
unused 100 mM DTT at the end of the day.
10X Reaction Buffer AM2
The reaction buffer is provided at a 10X concentration. It will be used as both a 1X and 10X solution
at different stages in the protocol. Please read the manual instructions carefully to ensure use of
the correct reaction buffer concentration.
To prepare a working solution of 1X Reaction Buffer, dilute 1:10 in dH2O and supplement with afinal concentration of 10 mM DTT. For example, for each 750,000 cell pellet add 40 µl 10X Reaction
Buffer AM2 to 320 µl dH2O plus 40 µl 100 mM DTT.
100X AdoMet
Is provided ready to use at a 100X concentration.

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GpC Methyltransferase Enzyme
The enzyme is provided ready to use at a concentration of 4 U/µl. Enough enzyme is supplied toperform 10 enzymatic treatments.
Stop Solution AM2
Is provided ready to use. Stop Solution AM2 may precipitate, therefore it is necessary to completely resuspend any precipitates by incubating at 37°C for 2 minutes and mixing prior to use.

Day 2 Buffer Preparation
RNase AIs provided ready to use at a concentration of 10 mg/ml.
Proteinase K
Is provided ready to use at a concentration of 10 mg/ml.
DNA Purification Binding Buffer (AM #103496)
Is provided ready to use.
DNA Purification Wash Buffer (AM #103497)
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.
3 M 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.
DNA Purification Elution Buffer (AM #103498)
Is provided ready to use.
Genomic DNA Purification Columns (AM #103945)
The kit includes 20 columns for 10 enzymatic treatments and 10 negative control reactions.
Conversion Buffer
Conversion Reagent is provided lyophilized. To make Conversion Buffer, resuspend the Conversion Reagent tube with 700 µl dH2O, 350 µl Buffer A and 175 µl Denaturation Reagent. Mix at room
temperature for 10 minutes with intermittent vortexing. The Conversion Buffer is a saturated
solution and it is normal for it not to dissolve completely. The Conversion buffer is stable for one
week at -20°C. Five vials of conversion reagent are provided. We suggest using each resuspended
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conversion reagent vial for a minimum of 4 samples.
Hydroquinone
Hydroquinone is an alkalizing agent and prevents DNA from stand breakage because of depurination. Prepare a working stock of Hydroquinone by resuspending one tube with 100 µl dH2O. Keepthe mixture in the dark and discard after use. Five vials of hydroquinone are provided. We suggest
using each resuspended hydroquinone vial for a minimum of 4 samples.

Day 3 Buffer Preparation
DNA Binding Buffer (AM #101232)
Is provided ready to use.
DNA Wash Buffer (AM #101233)
Is provided ready to use.
Desulfonation Buffer
For each reaction, combine 22 µl Buffer B with 88 µl dH2O and 110 µl 100% isopropanol.
DNA Elution Buffer (AM #100762)
Is provided ready to use.
DNA Purification Columns (AM #101037)
The kit includes enough columns for 20 bisulfite conversion desulfonation reactions and 20 PCR
amplification clean up reactions.
DNA Purification Binding Buffer (AM #103496)
Is provided ready to use.
3 M 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.
DNA Purification Wash Buffer (AM #103497)
The DNA Purification Wash Buffer should be ready to use following ethanol addition from firstuse.
DNA Purification Elution Buffer (AM #103498)
Is provided ready to use.

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12

Protocols – NOMe-Seq
Read the entire protocol before use.
Day 1

Approximate time frame for Day 1 is 6-8 hours. Stopping points areavailable after cell fixation and chromatin sonication if needed.

Step A: Cell Fixation1.

Prepare at least two 15 cm plates for each cell line to be tested. One plate will be used
to determine the cell count, while the remaining plate(s) will be used for the fixation andextraction. Grow the cells to 70-80% confluency. Stimulate cells as desired to activate thepathway of interest.

2.

When the plates are ready to harvest, use one 15 cm plate to perform a cell count. Trypsinize
the plate and count cells using a hemocytometer. This cell count is representative of thetotal number of cells to be fixed on the second 15 cm plate. The remaining cells can bediscarded after the count is completed.

3.

Prepare fresh fixation solutions for the remaining 15 cm plate(s). The volumes listed below areenough to process one 15 cm plate. Volumes may be scaled as indicated on page 9.

4.

a.

Fixation Solution: Add 0.54 ml 37% formaldehyde to 20 ml minimal cell culture
medium and mix thoroughly. Leave at room temperature.

b.

1X PBS Solution: Add 2.33 ml 10X PBS to 21 ml dH2O, mix and place on ice.

c.

Glycine Stop-Fix Solution: Combine 1 ml 10X Glycine Buffer, 1 ml 10X PBS and
8 ml dH2O. Mix well and leave at room temperature.

d.

Cell Scraping Solution: Add 0.6 ml 10X PBS to 5.4 ml dH2O, mix and place on ice.

Pour medium off the cells and add 20 ml Fixation Solution to each plate. Incubate on ashaking platform for 10 minutes at room temperature.
Note:

In standard protocols, chromatin is fixed for 10 minutes prior to shearing. Whilethese are typical fixation conditions, some protein/chromatin combinations may
work better with shorter fixation times (e.g. 5 minutes).

5.

Pour Fixation Solution off and wash by adding 10 ml ice-cold 1X PBS to each plate.
Rock the plate for 5 seconds, then pour off the PBS.

6.

Stop the fixation reaction by adding 10 ml Glycine Stop-Fix Solution to each of the plates.
Swirl to cover, and then rock at room temperature for 5 minutes.

7.

Wash each plate by pouring off the Glycine Stop-Fix Solution, then adding 10 ml ice-cold
1X PBS. Rock the plate for 5 seconds, then pour off the PBS.

8.

Just before use, add 30 µl 100 mM PMSF to Cell Scraping Solution. Add 5 ml of this ice-cold
Cell Scraping Solution to each plate and scrape cells with a rubber policeman. Hold the plateat an angle and scrape cells down to collect them at the bottom edge of the plate. Use a
1 ml pipette to transfer the cells to a 15 ml conical tube on ice.
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9.

Pellet the cells from Step A.8 by centrifugation for 10 minutes at 2,500 rpm (720 RCF) at 4°C.

10. Remove the supernatant and discard. At this point the protocol can be continued or the pellet can be frozen. If freezing the pellet, add 1 µl 100 mM PMSF and 1 µl PIC and freeze at -80°C.

Step B: Preparation of Sheared Chromatin by Sonication
1.

Thaw pellet (if necessary) on ice and resuspend cells in 1 ml ice-cold Lysis Buffer
supplemented with 5 µl PIC + 5 µl 100 mM PMSF. Incubate on ice for 30 minutes.

2.

(Optional) Transfer the cells to an ice-cold dounce homogenizer. Dounce on ice with 40
strokes to aid in nuclei release, pausing after every 10 strokes to ensure the chromatin does
not overheat. If not performing the dounce homogenization, proceed to step #3.
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.

3.

Transfer cells to a 1.5 ml microcentrifuge tube and centrifuge for 10 minutes at 5,000 rpm(2,400 RCF) in a 4°C microcentrifuge to pellet the nuclei.

4.

Carefully remove the supernatant and discard. Resuspend the nuclei pellet in 1 ml 1X NE Wash
Buffer and centrifuge for 10 minutes at 5,000 rpm (2,400 RCF) in a 4°C microcentrifuge topellet the nuclei.

5.

Carefully remove the supernatant and discard. Resuspend the nuclei pellet in 1 ml 1X NE Wash
Buffer and place on ice.

6.

Using the information obtained in Step A.2 for the total number of cells fixed per plate,determine the volume of cell suspension needed to obtain 750,000 cells. This cell number
is sufficient to perform two NOMe-Seq reactions (e.g. both a positive and negative control
reaction can be performed on the same sample). For additional NOMe-Seq reactions, create
multiple aliquots of 750,000 cells per tube. We do not recommend scaling cell numbers
at this stage as it will affect downstream sonication and enzymatic reaction steps. If more
sample is needed, please prepare multiple tubes.

		

Total number of fixed cells (Step A.2) / 1 ml NE Wash Buffer = Concentration cells/ml

		750,000 cells needed / Concentration = Volume (ml) required per tube

7.

Aliquot 750,000 cells per 1.5 ml microcentrifuge tube and centrifuge for 10 minutes at 5,000rpm (2,400 RCF) in a 4°C microcentrifuge. Any unused nuclei should be discarded.

8.

Prepare fresh dilutions of 100 mM DTT and 1X Reaction Buffer AM2 as described on page 10.

9.

Carefully remove the supernatant and discard. Resuspend each nuclei pellet in 300 µl 1XReaction Buffer AM2 and place on ice.
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1410. Sonicate the nuclei pellet(s) to a chromatin size of greater than 1 kb using a microtip probesonicator, such as Active Motif’s EpiShear™ Sonicator with a 1/8” probe (Catalog No. 53051) in
combination with an EpiShear Cooled Sonication Platform (Catalog No. 53073) to maintainprobe height and temperature consistency between samples.
Suggested settings: Sonicate on ice with a 25% amplitude, 30 seconds Sonicator “On” timeand 30 seconds “Off” for a total time of 3 minutes.
11.

Gently spin the tube to collect the liquid. Proceed directly to the GpC Methyltransferase
reaction.

Step C: Methyltransferase Reaction1.

Set up the GpC Methyltransferase reaction in a 1.5 ml microcentrifuge tube as described
below. We also recommend setting up a negative control reaction that does not include the
GpC Methyltransferase enzyme for determination of endogenous methylation.
Note:

Be sure to use the provided 10X Reaction Buffer AM2 and not the diluted buffer.
If the 100 mM DTT was prepared on the same day it can be used to set up the
methyltransferase reaction. Otherwise, prepare fresh 100 mM DTT.

Reagents

SampleNegative
Control

dH2O

32 µl

37 µl

10X Reaction Buffer AM2

5 µl

5 µl

100 mM DTT

5 µl

5 µl

100X AdoMet

3 µl

3 µl

Fragmented Chromatin (250,000 cells)

100 µl100 µlGpC Methyltransferase enzyme (4 U/µl)

5 µl

–

Total Volume

150 µl150 µl2.

Incubate the reactions at 37°C for 4 hours, adding 1 µl of 100X AdoMet per hour to eachreaction. Following each addition of AdoMet, mix tubes by inverting and return to 37°C.

3.

During the final hour of the incubation, place Stop Solution AM2 at 37°C to pre-warm.

4.

After the 4 hour incubation, stop the reaction by adding 50 µl of 37°C Stop Solution AM2 toeach tube.

5.

Reverse cross-links of the fixed chromatin by heating at 65°C overnight (~16 hrs).
Note:

Alternatively, reversal of cross-links can be accomplished by heating to 95°C for
15 minutes and then proceeding directly into Step D of the protocol.

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15

Day 2

Approximate time frame for Day 2 is 7-8 hours. Stopping points are available after DNA purification or bisulfite conversion if needed.

Step D: Proteinase K Treatment and DNA Purification
1.

Following reversal of the cross-links, return the tubes to room temperature and spin briefly
to collect any liquid inside the caps.

2.

Add 4 µl RNase A to each reaction and mix thoroughly. Incubate at room temperature for
30 minutes.

3.

Add 6 µl Proteinase K to each reaction and mix thoroughly. Incubate at 50°C for 1 hour.

4.

Briefly spin the tubes to collect the liquid to the bottom. Transfer each reaction to a new
1.5 ml microcentrifuge tube.

5.

Add 1050 µl (5x sample volume) DNA Purification Binding Buffer (AM #103496) to eachreaction. If needed, adjust the pH with 5 µl 3M Sodium Acetate as described below.
Note:

Check that the color of the DNA sample / DNA Purification Binding Buffer
mixture is bright yellow, not light orange or violet. If the color is light orange orviolet, this indicates that its pH is too high. Add more 3M Sodium Acetate
5 µl at a time, mixing after addition, until the color is bright yellow (see Figure1). This step is crucial to the successful binding and purification of your DNA
sample.
Figure 1: Solution color as a function of pH.
The DNA Purification Binding Buffer has a pH indicator dye,so that the pH of the solution can be easily determined.
Only apply the sample to the column when the solution isbright yellow (left), indicating a pH under 7.5. DNA will notbind to the column if the pH is higher than 7.5.
Note: A full-color PDF of this manual can be downloaded
from the Active Motif website, which will enable you to see
this figure in color.

6.

For each sample, place a Genomic DNA purification column (AM #103945) in the collection
tube and add each pH adjusted sample to its own column in 750 µl increments. Close the cap
on each column, place them with the collection tubes in a microcentrifuge and spin them at
14,000 rpm for 1 minute. Discard any flow through and repeat sample loading and centrifugation steps until no sample remains.

7.

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

8.

Prepare DNA Purification Wash Buffer (AM #103497) before the first use. Follow the instruction on page 11 for the addition of ethanol prior to using the solution. Add 750 µl DNA
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16

Purification Wash Buffer to each column and cap the column.
9.

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

10. Remove the column from the collection tube, then remove and discard the flow throughfrom the collection tube. Return the column to the collection tube.
11.

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.

12.

Transfer the column to a clean microcentrifuge tube.

13.

Add 30 µl of DNA Purification Elution Buffer (AM #103498) to the center of the column
matrix and incubate for 1 minute at room temperature.

14.

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

15.

Discard column and proceed to Step E below. Alternatively, purified DNA may be stored at-20C for future use.

Step E: Bisulfite Conversion
1.

Prepare the Conversion Buffer and Hydroquinone reagents as described on pages 11-12.

2.

In fresh 0.2 ml PCR tubes prepare the following reactions. The optimal range for bisulfite
conversion is 500 ng - 2 µg per reaction. The maximum volume of DNA that can be used per
reaction is 13 µl. Adjust the volume of DNA and water as needed to maintain a final volumeof 140 µl per reaction.
Reagents

SampleNegative
Control

GpC Methyltransferase treated DNA______ µl______ µldH2O______ µl______ µlHydroquinone

7 µl

7 µl

Conversion Buffer

120 µl120 µlTotal Volume

140 µl140 µl3.

Place the tubes in a thermocycler and set the program to start with an initial melt at 94°C for
3 minutes, then a 50°C conversion for 5 hours, followed by a hold at 4°C. To prevent evaporation of samples, use the heated lid or overlay reactions with mineral oil.

4.

Remove tubes and continue with Step F below. The protocol can be stopped here and DNA
samples can be kept at 4°C in the dark for up to 5 days.

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17

Day 3

Approximate time frame for Day 3 is 4 hours. Stopping points are available
after DNA purification or PCR amplification if needed.

Step F: On-Column Desulfonation and DNA Purification
1.

For each conversion reaction, aliquot 500 µl DNA Binding Buffer (AM #101232) into a 1.5 ml
microcentrifuge tube. Add the entire conversion reaction mixture to the DNA Binding Buffer
in the tube and vortex.

2.

Remove the desired number of DNA purification columns (AM #101037) and place each one ina collection tube.

3.

Pipet each DNA Binding Buffer/conversion reaction mixture into one of the DNA Purification
columns and spin at 10,000 rpm for 30 seconds in a microcentrifuge. Empty the collection
tube and place the column back on the collection tube.

4.

Add 200 µl DNA Wash Buffer (AM #101233) to each column and spin at 10,000 rpm for 30
seconds in a microcentrifuge.

5.

Prepare Desulfonation Buffer as described on page 12.

6.

Add 200 µl Desulfonation Buffer to each column and incubate 20 minutes at room temperature. Spin at 10,000 rpm for 30 seconds in a microcentrifuge. Empty the collection tube andplace the column back on the collection tube.

7.

Add 200 µl DNA Wash Buffer (AM #101233) to each column and spin at 10,000 rpm for
30 seconds in a microcentrifuge. Empty the collection tube and place the column back on
the collection tube.

8.

Repeat Step 7 to perform a second wash.

9.

Spin at 10,000 rpm for 1 minute in a microcentrifuge to remove any residual Wash Bufferfrom the column. Remove the column and place in a new 1.5 ml microcentrifuge tube.

10. Add 50 µl DNA Elution Buffer (AM #100762) directly to the filter of the column. Incubate
3 minutes at room temperature.
11.

Spin at 10,000 rpm for 30 seconds in a microcentrifuge. The eluate will contain the converted
DNA. DNA can be stored at -20°C until ready to use.

Step G: PCR Reactions to Amplify Genomic Region of Interest
1.

Prior to performing sequencing, it is recommended to PCR amplify the genomic region ofinterest. This requires the use of bisulfite-specific PCR primer sets that surround the GpC
methylated regions of interest. For Sanger sequencing, we suggest primer design such thatthe amplicon length falls between 400-600 bp. This will ensure a long enough sequencing read to confirm the location of nucleosomes, while still enabling reads by single-passsequencing.

2.

Please refer to page 8 for details on bisulfite specific primer design for PCR amplification.
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18

3.

4.

5.

Below is a suggested protocol for PCR amplification of bisulfite-treated DNA. Depending on
the design of the locus-specific primers and PCR mix being used, some optimization may
be required. We recommend using Bioline 2X MyTaq™ Master Mix for PCR reactions (Bioline
Catalog No. BIO-25041). First prepare a Master Mix for the PCR reactions. The volumes listed
in the chart are enough for 2 PCR reactions.
Reagents

PCR Master Mix(2 PCR rxns)

dH2O

52.5 µl2X MyTaq™ Mix

62.5 µl

Bisulfite-specific PCR primer mix (10 µM each)

5 µl

Total Volume

120 µl

In 0.2 ml PCR tubes, prepare the following PCR reactions.
Reagents

SampleNegative
Control

Bisulfite-treated DNA

2 µl

2 µl

PCR Master Mix

48 µl

48 µl

Total Volume

50 µl

50 µl

Mix tubes well and quick spin to collect liquid to the bottom of the PCR tube. Place tubes
in a PCR cycler and program the thermocycler as shown below. Modify the annealing
temperature as required for the locus-specific PCR primer mix. Optimal primer Tm should be
between 50°C-60°C. To prevent evaporation of samples, use the heated lid or overlay reactions with mineral oil.
95°C for 3 minutes(95°C for 30 sec., amplicon-dependent Tm for 30 sec., 72°C for 30 sec.) for 45 cycles
72°C for 4 minutes
Hold at 4°C

Step H: Purification of Amplified DNA
1.

Following DNA amplification, purify the DNA. If the amplified PCR product is a single amplicon, the PCR amplified product can be purified using the included DNA purification reagents.
However, if more than one PCR product is generated it is necessary to run the PCR reaction
on an agarose gel to excise the proper amplicon using a gel extraction and purification kit.

2.

For single amplicon DNA purification, use the DNA purification reagents provided:.

Add 250 µl (5x PCR sample volume) DNA Purification Binding Buffer (AM #103496) to eachwww.activemotif.com

19

reaction. If needed, adjust the pH of the solution with 5 µl 3M Sodium Acetate as described
below.
Note:

Check that the color of the DNA sample / DNA Purification Binding Buffer
mixture is bright yellow, not light orange or violet. If the color is light orange orviolet, this indicates that its pH is too high. Add more 3M Sodium Acetate
5 µl at a time, mixing after addition, until the color is bright yellow (see Figure1). This step is crucial to the successful binding and purification of your DNA
sample.
Figure 1: Solution color as a function of pH.
The DNA Purification Binding Buffer has a pH indicator dye,so that the pH of the solution can be easily determined.
Only apply the sample to the column when the solution isbright yellow (left), indicating a pH under 7.5. DNA will notbind to the column if the pH is higher than 7.5.
Note: A full-color PDF of this manual can be downloaded
from the Active Motif website, which will enable you to see
this figure in color.

3.

For each sample, place a DNA purification column (AM # 101037) in the collection tube andadd each pH adjusted sample to its own column. Close the cap on each column, place themwith the collection tubes in a microcentrifuge and spin them at 14,000 rpm for 1 minute.
Discard any flow through.

4.

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

5.

Use the previously prepared DNA Purification Wash Buffer (AM #103497). Add 750 µl DNA
Purification Wash Buffer to each column and cap the column.

6.

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

7.

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

8.

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.

9.

Transfer the column to a clean microcentrifuge tube.

10. Add 50 µl of DNA Purification Elution Buffer (AM #103498) to the center of the column
matrix and incubate for 1 minute at room temperature.
11.

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

12.

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

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20

Day 4+

Time frame will depend on the manufacturer’s instructions for thereagents used.

For locus-specific analysis of NOMe-Seq data, we recommend performing DNA cloning and Sanger
sequencing using at least 10 colonies per gene region according to the instructions in Step I. Better coverage can also be obtained when a larger number of clones are sequenced and analyzed.
Alternatively, to reduce the amount of labor and time required for analysis of a large collection ofgenes, it is possible to use Next-Gen sequencing on NOMe-Seq reactions. A protocol to prepare
bisulfite converted, PCR amplified DNA samples for Next-Gen sequencing is available on the Active Motif website at www.activemotif.com/nome-seq.

Step I: DNA Cloning and Sequencing
1.

The purified PCR product can be cloned into a DNA vector of choice according to the instructions of the manufacturer. If possible, plasmids containing the LacZ gene for blue/white
screening are recommended.

2.

Transform competent bacterial cells, such as Active Motifs RapidTrans TAMI (Catalog No.
11096), with 2 µl of ligation reaction. Follow the appropriate protocol for the competent cells
used. The RapidTrans™ TAMI chemically competent cell protocol is provided below as an
example.
a.

Remove the necessary number of chemically competent cell tubes from -80°C andplace on ice to thaw.

b.

Add 1-5 µl of ligated DNA vector to thawed cells. Mix by tapping tubes gently and replace on ice immediately. Do not mix by vortexing or pipetting. Do not add more than
5 µl (10% of competent cell volume) to each reaction.

c.

Incubate DNA and competent cells on ice for 30 minutes.

d.

Heat-shock the tubes by immersing in a 42°C water bath for exactly 30 seconds.

e.

Replace transformation reactions on ice for 2 minutes.

f.

Aseptically add 250 µl SOC medium to each reaction.

g.

Incubate tubes at 37°C for 1 hour with shaking at 225-250 rpm.

h.

Using a sterile spreader, plate out 20-200 µl of each transformation reaction on prewarmed LB agar plates with the appropriate antibiotic resistance for the cloning vector
used. If using blue/white screening, ensure the selective plate contains X-gal.

i.

Allow plates to completely absorb any excess mediaj.

Incubate inverted plates overnight at 37°C.

3.

Select 10 colonies (or more if desired) for screening. Select white colonies if using blue/white
screening as the expression of LacZ has been interrupted with the insertion of the ligation
product.

4.

Colonies can be screened by PCR to determine if they contain the proper insert prior to
submitting samples for sequencing. PCR reactions should be set up using the forward andwww.activemotif.com

21

reverse primers from the cloning vector for amplification of the insert. Prepare the PCR reactions as listed below. Prepare a PCR master mix containing everything except the template
DNA and aliquot 30 µl to individual PCR tubes. Keep on ice.

5.

Reagents

PCR Master Mix(10 PCR rxns)

dH2O

243.2 µl

10X PCR Buffer

35 µl

dNTPs (2 mM each)

35 µlCloning vector PCR primer mix (10 µM each)

35 µl

Taq DNA polymerase (5 U/µl)

1.8 µlTotal Volume

350 µlPick the colonies for analysis and create a screening plate using LB agar plates with theappropriate antibiotic resistance by touching a pipet tip from the original colony selection
onto the new screening plate and then place the tip directly into the appropriate PCR reaction. Pipet up and down to mix. Remove the pipet tip, and cap the PCR tubes. Place tubes ina thermocycler and amplify.
95°C for 3 minutes(95°C for 30 sec., amplicon-dependent Tm for 30 sec., 72°C for 75 sec.) for 35 cycles
72°C for 4 minutes
Hold at 4°C

6.

Following PCR amplification, run 2 µl of each PCR product on a 1% agarose gel to verify amplification. Confirm the amplicon is of the correct size. Proceed with plasmid DNA isolation andsequencing of correct colonies.

7.

Isolate plasmid DNA from the colonies with correct insert sizes on the screening plate using
any commercially available kit or published protocol.

8.

DNA isolated from the positive clones can be submitted for Sanger sequencing using primers
specific to the cloning vector of choice.

Section J: Data Analysis
Data analysis will require sequencing results to be evaluated separately for GpC and CpG methylation profiles. A comparison of the enzymatically treated sequencing results against the negative
control reference sequence results will resolve if methylation that occurs in a GpCpG trinucleotide
is the result of artificial or endogenous methylation.
The clarity of the methylation profile will depend on the number of GpC residues that cover aparticular region. The more GpC residues present, the better the resolution.
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1.

First plot the GpC methylation from the sequencing data. This information provides thedetails about nucleosome or transcription factor positioning.
a.

A region of GpC sequence data containing unmethylated cytosines spanning a distanceof 147 bp or larger can be identified as the location of a nucleosome.

b.

A region of GpC sequence data containing a range of unmethylated cytosines between
10-80 bp could be indicative of a protein binding site.

c.

Regions of methylated GpC dinucleotides indicate an area of the chromatin that isnucleosome depleted.

d.

Depending on the GpC density and quality of the sequencing read, sometime results
are not as clearly defined as stated in Steps a-c. Additional criteria are provided to help
with interpretation of the data.

•

3 or more consecutively methylated GpC sites represent a nucleosome depleted region.

•

If one unmethylated GpC site exists within a stretch of methylated GpCs, the region
can still be considered nucleosome depleted. The unmethylated site may represent a
protein binding site.

•

If one methylated GpC site exists within a stretch of unmethylated GpCs, the region
may still represent a nucleosome occupied sequence.

2.

Second, plot the CpG methylation from the sequencing data. This information provides
details about the endogenous DNA methylation present at the region of interest.

3.

Manual methods will have to be used to overlay the two methylation profiles together
in order to visualize the temporal relationship between nucleosome occupancy and DNA
methylation.

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23

References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.

You, J.S. et al. (2011) Proc. Natl. Acad. Sci., 108: 14497-14502.
Wolff, E.M. et al. (2010) PLoS Genetics, 6(4): e1000917.
Kelly, T.K. et al. (2010) Mol. Cell, 39: 901-911.
Kelly, T.K. et al. (2012) Genome Res., doi:10.1101/gr.143008.112.
Jenuwein, T. and Allis, C.D. (2001) Science, 293: 1074-1080.
Lin, J.C. et al. (2007) Cancer Cell, 12: 432-444.
Boeger, H. et al. (2003) Mol. Cell, 11: 1587-1598.
Boeger, H. et al. (2004) Mol. Cell, 14: 667-673.
Lusser, A. and Kadonaga, J.T. (2003) Bioessays, 25: 1192-1200.
Studitsky, V.M. et al. (2004) Trends Biochem. Sci., 29: 127-135.
Yuan, G.C. et al. (2005) Science, 309: 626-630.
Zhu, W.G. et al. (2003) Mol. Cell Biol., 23: 4056-4065.
Langst, G. and Becker, P.B. (2004) Biochim. Biophys. Acta., 1677: 58-63.
Fragoso, G. et al. (1995) Genes Dev., 9: 1933-1947.
Bouazoune, K. et al. (2009) Nucl. Acids Res., 37: 5279-5294.
Giresi, P.G. and Lieb, J.D. (2009) Methods, 48: 233-239.
Xu, M. et al. (1998) Nucl. Acids Res., 26(17): 3961-3966.
Kilgore, J.A. et al. (2007) Methods, 41: 320-332.
Pardo, C.E. et al. (2011) Nucl. Acids Res., 39(1) e5, doi:10.1093/nar/gkq716.

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24

Appendix
Section K: Troubleshooting Guide
Problem/question

Recommendation

Poor yield of shearedchromatin

Nuclei not released. It is highly recommended to perform dounce homogenization. Usea dounce homogenizer with a small clearance pestle (Catalog No. 40401). Monitor cell
lysis under a microscope. Generally, the more cells that are lysed, the higher the shearedchromatin yield.
Decrease the fixation time. Over-fixed cells are often very resistant to lysis and shearing.
Cross-linking for longer periods of time tends to cause cells to form into a giant crosslinked aggregate that is not sheared efficiently. Decrease the incubation time of theformaldehyde fixation step to 5 minutes.
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 Fixation Solution.
Buffers were not scaled proportionally to the size of the sample. Use the chart on page 9
to scale up or down chromatin preparation.

Low amount of GpC
Methyltransferase activity

Confirm that all the reagents were added to the methyltransferase reaction including GpC
enzyme and AdoMet and that reactions were performed at the temperatures indicatedin the protocol. AdoMet should be spiked into the reaction every hour during the 4 hour
incubation.
Do not exceed the recommendations of 250,000 cell equivalents per reaction. The
amount of enzyme is optimized for use in this range and cannot be scaled for use withdifferent cell equivalent.

Poor bisulfite conversion

We recommend using a DNA starting volume of 13 µl. However, volumes up to 20 µl canbe used without affecting the protocol.
A 5 hour conversion incubation at 50°C is normally sufficient to efficiently convert DNA
samples. However, DNA sequences that are GC-rich and/or contain extensive secondary
structures may require a 9 hour conversion at 50°C. If noticing a loss of DNA after theconversion reaction, the conversion time can be reduced to as little as 3 hours.
The Conversion Reagent is sensitive to air and moisture. We recommend performing
bisulfite conversion on 4 samples per experiment to utilize a single tube of the supplied
Conversion Reagent. Unused Conversion Reagent that is in solution should be discarded
at the end of the day and not stored for future use.
Hydroquinone solution is not stable long term. We recommend performing bisulfite
conversion on 4 samples per experiment to utilize a single tube of the supplied Hydroquinone. Unused Hydroquinone that is in solution should be discarded at the end of the day
and not stored for future use.

Storage of DNA

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Once DNA is bisulfite converted samples may be stored at -20°C prior to PCR analysis.
However, we recommend heating the frozen material to 37°C for 10 minutes before use in
PCR, as heat-treatment releases any DNA bound to the tube during storage.

25

Problem/question

Recommendation

No amplification of DNA
by PCR

Follow the recommendations for PCR primer design on page 8 to prepare bisulfite
converted primers for your gene region of interest.
PCR conditions may need to be optimized based on the annealing temperature of thegene-specific PCR primers, the Master Mix and PCR instrument used. We have validated
the kit with Bioline 2X MyTaq™ Master Mix (Catalog No. BIO-25041).
The pH of the Binding Buffer for the DNA purification step was too high. Your sample
should be bright yellow when loading onto the DNA purification column. If the color islight orange or violet, 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 andpurification. For a full color image please see the manual for Active Motif’s Chromatin IP
DNA Purification Kit Catalog No. 58002 available online at www.activemotif.com.
No ethanol in the DNA Purification Wash Buffer. Make sure that ethanol has been added
to the DNA Purification Wash Buffer (AM # 103497) prior to first use.
DNA will not be eluted from the silica matrix of the DNA purification column if the bufferused for elution contains high salt. Use the included DNA Purification Elution Buffer (AM #103498) for DNA purification following reversal of chromatin cross-links and DNA purification of amplified locus-specific PCR products.

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26Section L. Related Products
ChIP-IT® Kits		

FormatCatalog No.

ChIP-IT® High Sensitivity		
ChIP-IT® Express		ChIP-IT® Express Enzymatic		
ChIP-IT® Express Shearing Kit		
ChIP-IT® Express Enzymatic Shearing Kit		
ChIP-IT® Express HT		
Re-ChIP-IT®		RNA ChIP-IT®		
Chromatin IP DNA Purification Kit		
EpiShear™ Multi-Sample Sonicator		
EpiShear™ Probe Sonicator		ChIP-IT® Protein G Magnetic Beads		
Siliconized Tubes, 1.7 ml		
ChIP-IT® qPCR Analysis Kit		
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		

16 rxns
25 rxns
25 rxns
10 rxns
10 rxns
96 rxns
25 rxns
25 rxns
50 rxns
110 V
110 V
25 rxns
25 tubes
10 rxns
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

53040
53008
53009
53032
53035
53018
53016
53024
58002
53062
53051
53014
53036
53029
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.
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

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
Toll Free:877 222 9543
Telephone: 760 431 1263
Fax:
760 431 1351
E-mail:tech_service@activemotif.com
Active Motif Europe
Avenue Reine Astrid, 92
B-1310 La Hulpe, Belgium
UK Free Phone: 0800 169 31 47
France Free Phone:
0800 90 99 79
Germany Free Phone: 0800 181 99 10
Telephone:		+32 (0)2 653 0001
Fax:		
+32 (0)2 653 0050
E-mail: 		
eurotech@activemotif.com
Active Motif Japan
Azuma Bldg, 7th Floor
2-21 Ageba-Cho, Shinjuku-Ku
Tokyo, 162-0824, Japan
Telephone: +81 3 5225 3638
Fax:
+81 3 5261 8733
E-mail:japantech@activemotif.com
Visit Active Motif on the worldwide web at http://www.activemotif.com
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
Don’t forget to bookmark our site for easy reference!
www.activemotif.com

28