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SimpleDIP™ Hydroxymethylated DNA IP (hMeDIP) Protocol

Required Reagents

Reagents Included

No Name
31482 SimpleDIP™ Cell Lysis Buffer
49291 SimpleDIP™ DNA IP Buffer (10X)
7009 ChIP Elution Buffer (2X)
74252 TE Buffer
89173 3M Sodium Acetate, pH 5.2
9006 ChIP-Grade Protein G Magnetic Beads
10007 DNA Binding Buffer
10008 DNA Wash Buffer (add 4x volume ethanol before use)
10009 DNA Elution Buffer
10010 DNA Purification Columns
10012 Proteinase K
7013 RNase A
51660 5-Hydroxymethylcytosine (5-hmC) (HMC31) Mouse mAb
98528 Mouse (G3A1) mAb IgG1 Isotype Control (DIP Formulated)
86179 SimpleDIP™ Hydroxymethyl Control Spike-In DNA
20906 SimpleDIP™ Hydroxymethyl Control Primers

Reagents Not Supplied

No Name
7017 / 14654 Magnetic Separation Rack
9872 Phosphate Buffered Saline (PBS-1X) pH7.2 (Sterile)
12931 Nuclease-free water
- Phenol/Choloroform/Isoamyl Alcohol (25:24:1) Saturated with 10 mM Tris, pH 8.0, 1 mM EDTA
- Chloroform:Isoamyl Alcohol (24:1)
- Ethanol (96-100%)
- Trypsin
- Taq DNA polymerase
- dNTP mix
- Real-Time PCR SYBR™ Green Reaction Mix

Section I. Genomic DNA Extraction

Before Starting

  • Stimulate or treat 5 million cells for each experiment. This number of cells will generate approximately 30 μg of DNA (30 IPs).
  • Remove and warm SimpleDIP™ Cell Lysis Buffer in a 37°C water bath and ensure SDS is in solution.
Number of Cells Used Approximate Yield
1 million 6 μg
5 million 30 μg
10 million 60 μg
    1. For suspension cells, count cells using a hemocytometer.

    2. For adherent cells, remove media and wash cells with 10 ml ice-cold 1X PBS, completely removing wash from culture dish. Add 2 ml of trypsin to remove the cells from the plate. Add 8 ml of media with serum to neutralize the trypsin after cells are completely detached and mix thoroughly. Count cells using a hemocytometer.

  1. Transfer 5 million cells to a 15 ml conical tube, centrifuge at 250 x g in a bench top centrifuge for 5 min at 4°C. Wash pellet twice with 10 ml ice-cold 1x PBS. Repeat centrifugation after each wash.
  2. Resuspend the cell pellet from step 2 in 500 μl of SimpleDIP™ Cell Lysis Buffer.
  3. Transfer cells and buffer into a 1.5 ml microcentrifuge tube. Add 2 μl of Proteinase K to the tube and incubate overnight (12-18 hr) with shaking at 55°C.
  4. Add 500 μl of phenol/chloroform/isoamyl alcohol (25:24:1) and mix thoroughly by vortexing for 30 sec.
  5. Separate layers by centrifugation at 10,000 x g for 5 min in a microcentrifuge. Carefully transfer the top aqueous layer to a new tube.
  6. Add 500 μl of chloroform/isoamyl alcohol (24:1) to the material and mix thoroughly by vortexing for 30 sec.
  7. Separate layers by centrifugation at 10,000 x g for 5 min in a microcentrifuge. Carefully transfer the top aqueous layer to a new tube.
  8. Add 50 μl of 3M Sodium Acetate, pH 5.2, then 1.0 ml of 100% ethanol chilled at -20°C. Incubate at -20°C overnight or -80°C for 1 hr to precipitate DNA.
  9. Pellet DNA by centrifugation at 10,000 x g for 5 min in a microcentrifuge.
  10. Carefully remove supernatant and wash pellet with 70% ethanol chilled at -20°C. Decant supernatant and air dry or vacuum dry pellet.
  11. Resuspend pellet in 500 μl of TE Buffer and add 2 μl of RNase A. Incubate for 30 min at 37°C.
  12. Repeat steps 5-11 and then resuspend pellet in 200 μl TE Buffer.

Section II. Genomic DNA Shearing and Quantification

  1. Sonicate genomic DNA (from Section I, Step 13) for 5 pulses for 15 sec each at medium setting, keeping tube on ice for 30 sec in between each pulse.

    • Genomic DNA from mouse embryonic stem cells was fragmented to sub 500 bp with 5 sets of 15 sec pulses using a VirTis VIRSONIC 100 Ultrasonic Homogenizer/Sonicator (The VirTis Company, Gardiner, NY) at setting 6 with a 1/8-inch probe.

    • Please see Appendix A for further optimization of sonication conditions.

  2. Remove 5 μl of the genomic DNA from Step 1 and determine DNA fragment size by electrophoresis on a 1% agarose gel with a 100 bp DNA marker. DNA should be sheared to a length of approximately 100-500 bp.
  3. To determine DNA concentration, transfer 2 μl of genomic DNA from Step 1 to 98 μl TE Buffer to give a 50-fold dilution and read the OD260. The concentration of DNA in μg/ml is OD260 x 2,500. DNA concentration should ideally be between 50 and 150 μg/ml.

Section III. DNA Immunoprecipitation

NOTE: The 5-Hydroxymethylcytosine (5-hmC) (HMC31) Mouse mAb binds hydroxymethylated genomic DNA only in the context of single-stranded DNA. However, next-generation sequencing library prep kits require double-stranded DNA for the adaptor ligation step and won’t work efficiently with the heat-denatured DNA from the hMeDIP protocol. Therefore, before setting up the DNA immunoprecipitation, the user must perform the adaptor ligation step as recommended by the manufacturer’s DNA library preparation protocol. The user should then use 1 ug of adaptor-ligated DNA for the DNA immunoprecipitation.

Before Starting

  • Remove and warm 10X SimpleDIP™ IP Buffer in a 37°C water bath and ensure there is no precipitate.
  1. In one tube, prepare enough IP mix for the desired number of IPs (see table below). When determining the number of IPs, the user should include the negative control Mouse (G3A1) mAb IgG1 Isotype Control (DIP Formulated) #98528 sample and 1 additional IP to account for the 10% input. Place mix on ice.
Reagent Amount per IP/Input
10x SimpleDIP™ DNA IP Buffer 50 μl
Sonicated genomic DNA 1 μg
SimpleDIP™ Hydroxymethyl Control Spike-In DNA (optional) 1 μl
dH2O Up to 500 μl final volume
  1. Remove a 50 μl sample of the diluted DNA and transfer to a microfuge tube. This is the 10% input sample.
  2. For each IP, transfer 500 μl of IP mix to a 1.5 ml microcentrifuge tube and heat each tube for 10 min at 95°C to denature DNA. Be sure to also heat the 10% input. Quickly put samples on an ice water bath for 5 min.

    • From this point forward, it is important to keep all buffers cold and keep samples on ice to maintain single stranded DNA. The input can now be stored at -20°C until further use.

  3. Add 10 μl of 5-Hydroxymethylcytosine (5-hmC) (HMC31) Mouse mAb #51660 or 10 μl of Mouse (G3A1) mAb IgG1 Isotype Control (DIP Formulated) #98528 to the appropriate IP samples. Incubate samples overnight at 4°C with rotation.
  4. Resuspend ChIP Grade Protein G Magnetic Beads #9006 by gently vortexing. Immediately add 20 μl of ChIP Grade Protein G Magnetic Beads #9006 to each sample and incubate for 2 hours at 4°C with rotation. Proceed to Section IV.

Section IV. Washing and Elution of the Immunoprecipitated DNA

  • Before starting prepare and chill on ice for each IP:
  • 4 ml 1X SimpleDIP™ DNA-IP Buffer (400 μl 10X SimpleDIP™ IP Buffer + 3.6 ml water)
  • Remove and warm 2X ChIP Elution Buffer in a 37°C water bath and ensure SDS is in solution.
  • Prepare 150 μl 1X ChIP Elution Buffer (75 μl 2x ChIP Elution Buffer + 75 μl water) for each IP and 10% input sample.
  • Set a water bath or thermomixer to 65°C.
  1. Pellet protein G magnetic beads (from Section III, Step 5) by placing the tubes in a magnetic separation rack. Wait 1 to 2 min for solution to clear and then carefully remove the supernatant.
  2. Add 1 ml of 1X SimpleDIP™ DNA IP buffer to the beads and incubate at 4°C for 5 min with rotation.
  3. Pellet protein G magnetic beads by placing the tubes in a magnetic separation Rack. Wait 1 to 2 min for solution to clear and then carefully remove the supernatant.
  4. Repeat Steps 2 and 3 three additional times, for a total of 4 washes.
  5. Add 150 μl of the 1X ChIP Elution Buffer to each IP sample, including the 10% input sample tube. Set aside input sample at room temperature until Step 9.
  6. Elute DNA from the antibody/protein G beads for 30 min at 65°C with gentle vortexing (1,200 rpm). A thermomixer works best for this step. Alternatively elutions can be performed at room temperature with rotation, but may not be as complete.
  7. Pellet protein G magnetic beads by placing the tubes in a Magnetic Separation Rack and wait 1 to 2 min for solution to clear.
  8. Carefully transfer eluted DNA to a new tube.
  9. Immediately proceed to Section V. Alternatively, samples can be stored -20°C. However, to avoid formation of a precipitate, be sure to warm samples to room temperature before adding DNA Binding Buffer (Section V, Step 1).

Section V. DNA Purification using Spin Columns

Before Starting

  • Add 10 ml of ethanol (96-100%) to DNA Wash Buffer before use. This step only has to be performed once prior to the first set of DNA purifications.
  • Remove one DNA spin column/collection tube for each DNA sample from Section IV.
  1. Add 750 μl DNA Binding Buffer to each DNA sample and vortex briefly.

    • 5 volumes of DNA Binding Buffer should be used for every 1 volume of sample.

  2. Transfer 450 μl of each sample from Step 1 to a DNA Purification Column in a collection tube.
  3. Centrifuge at 18,500 x g in a microcentrifuge for 30 sec.
  4. Remove the spin column from the collection tube and discard the liquid. Replace spin column in the collection tube.
  5. Transfer the remaining 450 μl of sample from Step 1 to the spin column in collection tube and repeat steps 3 and 4.
  6. Add 750 μl of DNA Wash Buffer to the spin column in collection tube.
  7. Centrifuge at 18,500 x g in a microcentrifuge for 30 sec.
  8. Remove the DNA spin column from the collection tube and discard the liquid. Replace spin column in the collection tube.
  9. Centrifuge at 18,500 x g in a microcentrifuge for 30 sec.
  10. Discard collection tube and liquid. Retain spin column.
  11. Add 50 μl of DNA Elution Buffer to each DNA spin column and place into a clean 1.5 ml microcentrifuge tube.
  12. Centrifuge at 18,500 x g in a microcentrifuge for 30 sec to elute DNA.
  13. Remove and discard DNA spin column. Eluate is now purified DNA. Samples can be stored at -20°C.

Section VI. Quantification of DNA by PCR

Recommendations

  • Use filter-tip pipette tips to minimize risk of contamination.
  • The control primers included in the kit are specific for the SimpleDIP™ Hydroxymethyl Control Spike-In DNA #86179 and can be used for standard PCR or quantitative real-time PCR.
  • A hot-start Taq polymerase is recommended to minimize the risk of non-specific PCR products.
  • PCR primer selection is critical. Primers should be designed with close adherence to the following criteria:
Primer length 24 nucleotides
Optimum Tm 60°C
Optimum GC 50%
Amplicon size 150 to 200 bp (for standard PCR) 80 to 160 bp (for real-time quantitative PCR)

Standard PCR Method:

  1. Label the appropriate number of 0.2 ml PCR tubes for the number of samples to be analyzed. These should include the 10% input sample and the negative control Mouse (G3A1) mAb IgG1 Isotype Control (DIP Formulated) #98528 and a tube with no DNA to control for DNA contamination.
  2. Add 2 μl of the appropriate DNA sample to each tube.
  3. Prepare a master reaction mix as described below, making sure to add enough reagent for two extra tubes to account for loss of volume. Add 18 μl of master mix to each reaction tube.
Reagent Volume for 1 PCR Reaction (18 μl)
Nuclease-free dH2O 12.5 μl
10x PCR Buffer 2.0 μl
4 mM dNTP Mix 1.0 μl
5 μM Primers 2.0 μl
Taq DNA Polymerase 0.5 μl
  1. Start the following PCR reaction program:
a. Initial Denaturation 95°C 5 min
b. Denature 95°C 30 sec
c. Anneal 62°C 30 sec
d. Extension 72°C 30 sec
e. Repeat Steps b-d for a total of 34 cycles.
f. Final Extension 72°C 5 min
  1. Remove 10 μl of each PCR product for analysis by 2% agarose gel or 10% polyacrylamide gel electrophoresis with a 100 bp DNA marker. The expected size of the PCR product for the postive control spike in #20906 is 120 bp.

Real-Time Quantitative PCR Method:

  1. Label the appropriate number of PCR tubes or PCR plates compatible with the model of PCR machine to be used. PCR reactions should include the negative control Mouse (G3A1) mAb IgG1 Isotype Control (DIP Formulated) #98528, a tube or well with no DNA to control for contamination, and a serial dilution of the 10% input genomic DNA (undiluted, 1:5, 1:25, 1:125) to create a standard curve and determine the efficiency of amplification.
  2. Add 2 μl of the appropriate DNA sample to each tube or well of the PCR plate.
  3. Prepare a master reaction mix as described below. Add enough reagents for two extra reactions to account for loss of volume. Add 18 μl of reaction mix to each PCR reaction tube or well.
Reagent Volume for 1 PCR Reaction (18 μl)
Nuclease-free H2O 6 μl
5 μM primers 2 μl
SYBR™ Green Reaction Mix 10 μl
  1. Start the following PCR Reaction program:
a. Initial Denaturation 95°C 3 min
b. Denature 95°C 15 sec
c. Anneal 65°C 60 sec
d. Repeat step b and c for a total of 40 cycles
  1. Analyze quantitative PCR results using the software provided with the real-time PCR machine. Alternatively, one can calculate the IP efficiency manually using the Percent Input Method and the equation shown below. With this method, signals obtained from each immunoprecipitation are expressed as a percent of the total input chromatin.

    Percent Input = 10% x 2(C[T] 10% Input Sample – C[T] IP Sample)
    C[T] = CT = Threshold cycle of PCR reaction

Appendix A. Optimization of Sonication Conditions

Optimal conditions for shearing genomic DNA to 150-500 bp in length may depend on cell type and number of cells and the type of sonicator used. Below is a protocol to determine the optimal sonication conditions for a specific cell type and concentration of cells.

  1. Prepare genomic DNA from 5 million cells as described in Section I, Steps 1-13.
  2. Sonicate on medium setting for 10 pulses for 15 sec each, keeping tube on ice for 30 sec in between each pulse. Take a 5 μl aliquot after every 2 pulses. Determine DNA fragment size by electrophoresis in a 1% agarose gel with a 100 bp DNA marker.
  3. Observe which of the sonication conditions produces DNA in the desired range of 150-500 bp. These conditions can then be used in Section II, Step 1 in place of the suggested protocol step.
Genomic DNA from 5 million mouse ES cells

Genomic DNA from 5 million mouse ES cells was fragmented with 0, 2, 4, 6, 8, and 10 sets of 15 sec pulses using a VirTis Virsonic 100 Ultrasonic Homogenizer/Sonicator at setting 6 with a 1/8-inch probe. DNA samples were then separated by electrophoresis on a 1% agarose gel next to a 100 bp ladder and stained with ethidium bromide.

Appendix B. Troubleshooting Guide

Protocol Step Issue Causes and Resolutions
Protocol Step Issue Causes and Resolutions
Genomic DNA Extraction Concentration of fragmented DNA is too low. Not enough cells were added to the genomic DNA extraction. Count a separate plate of cells before performing the genomic DNA extraction to ensure an exact count. The genomic DNA extraction protocol can support up to 10 million cells per 500 ml of SimpleDIP™ Cell Lysis Buffer. Adding more than 10 million cells may inhibit cell lysis and also decrease DNA concentration.
Genomic DNA Shearing and Quantification OD260/280 ratio is lower than 1.8 (impure DNA). Phenol and/or salt carryover occurred during the phenol/chloroform extractions. During the extractions, leave a small amount of the top layer behind ensuring that no phenol or salt is accidentally transferred with the DNA-containing supernatant.
DNA fragments are the incorrect size. Sonication power or the number of pulses was not sufficient to shear the DNA properly. See Appendix A for a DNA shearing optimization protocol.
DNA Immunoprecipitation Can I alter the amount of antibody or DNA used in the IP? The kit was optimized for 1 μg of antibody and 1 μg of genomic DNA. Adding less antibody or DNA may decrease the recovery of hydroxymethylated DNA, while adding additional antibody or DNA may decrease the specificity of the IP and generate false positive enrichments.
Can other antibodies be used in the kit? The protocol has been validated and optimized for use with the antibody included in the kit. Other antibodies may not perform optimally with the protocol provided in the kit.
Quantification of DNA by PCR Little or no enrichment of hydroxymethylated DNA In each IP, 1 μg of antibody and 1 μg of genomic DNA should be used. Using less of either may result in decreased recovery of hydroxymethylated DNA and weaker signal.
The antibody will only bind to single-stranded DNA, so ensure that all protocol steps after denaturation are performed on ice to prevent reannealing.
Incomplete elution of DNA from the beads may decrease recovery of hydroxymethylated methylated DNA and result in weaker signal. Elution of DNA from protein G beads is optimal at 65°C with frequent mixing to keep beads suspended in solution.
High background in the IgG control immunoprecipitation. In each IP, 1 μg of antibody and 1 μg of genomic DNA should be used. Using additional antibody or DNA may generate higher background by increasing the amount of non-specific interactions. Adding less DNA could cause the signal in the IgG PCR reaction to appear higher relative to your input.
If performing gel-based PCR, scale back on the number of cycles to be sure you are analyzing PCR products within the linear amplification phase of PCR. Otherwise the differences in quantitites of starting DNA cannot be accurately measured. Alternatively, quantify your immunoprecipitations by real-time PCR.
DIP-Sequencing Can this kit be used in sequencing? Yes. However, next-generation sequencing library prep kits require double-stranded DNA for the adaptor ligation step and won’t work efficiently with enriched heat-denatured DNA from the MeDIP protocol. Therefore, before setting up the DNA immunoprecipitation in Section III, the user must perform the adaptor ligation step as recommended by the manufacturer’s DNA library preparation protocol. The user should then use 1 μg of adaptor-ligated DNA for the DNA immunoprecipitation.
Storage When can the protocol be stopped and the material stored until the protocol is ready to be finished? Cell pellets can be flash frozen and stored at -80°C after Section I, Step 2.
Genomic DNA can be stored at -20°C after Section I, Step 9 or Step 13.
Sheared DNA can be stored at -20°C after Section II, Step 3.
DNA IP’s can be stored at -20°C overnight after Section IV, Step 9. However, to avoid formation of precipitate, be sure to warm samples to room temperature before adding DNA Binding Reagent A in Section V, Step1.
IP’d genomic DNA can be stored at -20°C after Section V, Step 13. However, be sure to heat frozen material to 37°C for 10 minutes before use in PCR, as heat treatment releases any DNA bound to the tube during storage.

posted November 2015

Product Includes Volume (with Count) Storage Temp
SimpleDIP™ Cell Lysis Buffer 1 x 6 ml 4°C
SimpleDIP™ DNA-IP Buffer (10X) 1 x 5 ml 4°C
ChIP Elution Buffer (2X) 1 x 1.5 ml 4°C
TE Buffer 1 x 10 ml 4°C
3 M Sodium Acetate, pH 5.2 1 x 1.2 ml 4°C
ChIP-Grade Protein G Magnetic Beads 9006 1 x 200 µl 4°C
DNA Binding Buffer 1 x 12 ml RT
DNA Wash Buffer (add 4x volume ethanol before use) 1 x 2.5 ml RT
DNA Elution Buffer 1 x 1 ml RT
DNA Purification Columns and Collection Tubes 1 x 15 Pack RT
Proteinase K 1 x 20 µl -20°C
RNAse A (10 mg/ml) 1 x 50 µl -20°C
5-Hydroxymethylcytosine (5-hmC) (HMC31) Mouse mAb 51660 1 x 100 µl -20°C
Mouse (G3A1) mAb IgG1 Isotype Control (DIP Formulated) 1 x 100 µl -20°C
SimpleDIP™ Hydroxymethyl Control Spike-In DNA 1 x 20 µl -20°C
SimpleDIP™ Hydroxymethyl Control Primers 1 x 150 µl -20°C

Product Description

The SimpleDIP™ Hydroxymethylated DNA IP (hMeDIP) Kit provides enough reagents to perform up to 10 genomic DNA preparations and 10 IPs from mammalian cells and is optimized for 1 μg of genomic DNA per IP. The SimpleDIP™ protocol can be performed in as little as two days and can easily be scaled up or down for use with more or less cells. Cells are first lysed and genomic DNA is extracted and sonicated into small fragments (200-500 bp). DNA IPs are performed using 5-Hydroxymethylcytosine (5-hmC) (HMC31) Mouse mAb and ChIP-Grade Protein G Magnetic Beads. After elution from the beads, the DNA is purified using DNA purification spin columns provided in the kit. The enrichment of particular DNA sequences can be analyzed by a variety of methods including standard PCR, quantitative real-time PCR, or next-generation sequencing. The SimpleDIP™ 5-Hydroxymethylcytosine DNA IP Kit provides a highly validated 5-hmC monoclonal antibody to ensure specific and robust signal. The kit also includes DNA that contains exclusively 5-hydroxymethylcytosine, which can be spiked-in to the IPs as a control. Thus, spiked-in DNA will be immunoprecipitated with 5-Hydroxymethylcytosine (HMC1) Mouse mAb, but not with the Mouse (G3A1) mAb IgG1 Isotype Control (DIP Formulated). The relative enrichment can then be quantified using the SimpleDIP Hydroxymethyl Control Primers.


Specificity / Sensitivity

The SimpleDIP™ Hydroxymethylated DNA IP (hMedIP) Kit can be utilized to detect endogenous levels of 5-hydroxymethylcytosine modifications in mammalian cells (see Figure 1). The 5-Hydroxymethylcytosine (5-hmC) (HMC31) Mouse mAb has been validated for specificity using ELISA, dot blot and hMeDIP assays and shows high specificity for its target DNA modification (see Figures 2-4). A positive control IP spike-in DNA fragment containing 5-hydroxymethylcytosine and positive control primer set for amplification of this fragment are included in the kit. This spike-in DNA and primer set can be used as a positive control for IP with any mammalian cell type.


DNA immunoprecipitation (DIP) is a technique that uses antibodies to immunoenrich for regions of the genome containing modified nucleotides. This assay was first used with a 5-methylcytosine antibody to identify differentially methylated sites within normal and transformed cells (1). Investigators can use the DIP assay to look at specific genomic loci or look across the entire genome by utilizing next-generation sequencing (NGS) (2). When performing the DIP assay, cells are first lysed and the nucleic acids are recovered using phenol-chloroform extraction and ethanol precipitation. RNA is then removed by RNase A digestion, and genomic DNA is isolated by a second round of phenol-chloroform extraction and ethanol precipitation. The resulting genomic DNA is then fragmented by either restriction enzyme digestion or sonication and subjected to immunoprecipitation (IP) using antibodies specific to the modified nucleotide. Any sequences containing the modified nucleotide will be enriched by the immunoselection process. After IP, the DNA is purified and Quantitative Real-Time PCR can be used to measure the amount of enrichment of a particular DNA sequence. Alternatively, the DIP assay can be combined with NGS to provide genome-wide analysis of a specific DNA modification.


1.  Weber, M. et al. (2005) Nat Genet 37, 853-62.

2.  Down, T.A. et al. (2008) Nat Biotechnol 26, 779-85.



For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
SimpleChIP is a registered trademark of Cell Signaling Technology, Inc.
SimpleDIP is a trademark of Cell Signaling Technology, Inc.
SYBR is a registered trademark of Life Technologies Corporation.

95176
SimpleDIP™ Hydroxymethylated DNA IP (hMeDIP) Kit