FIGURE 1. DNA immunoprecipitations were performed with 1 μg of genomic DNA from mES cells and either 10 μl of 5-Hydroxymethylcytosine (5-hmc) (HMC31) Mouse mAb #51660 or 10 μl of Mouse (G3A1) mAb IgG1 Isotype Control (DIP Formulated) using SimpleDIP™ Hydroxymethylated DNA IP (hMeDIP) Kit #95176. The enriched DNA was quantified by real-time PCR using mouse Aqp2 exon 1 primers, SimpleDIP™ Mouse Intracisternal-A Particle (IAP) LTR Primers, mouse Lamc3 exon 1 primers, and SimpleChIP® Mouse GAPDH Intron 2 Primers #8986. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input DNA, which is equivalent to one.
FIGURE 2. 5-Hydroxymethylcytosine (5-hmC) (HMC31) Mouse mAb #51660 specificity was determined by dot blot. The same sequence of a 387 base pair DNA fragment was generated by PCR using exclusively unmodified cytosine, 5-methylcytosine (5-mC), 5-hydroxymethylcytosine (5-hmC), 5-carboxylcytosine (5-caC), or 5-formylcytosine (5-fC). The respective DNA fragments were blotted onto a nylon membrane, UV cross-linked, and probed with 5-Hydroxymethylcytosine (5-hmC) (HMC31) Mouse mAb. The top panel shows the antibody only binding to the DNA fragment containing 5-hmC, while the bottom panel shows the membrane stained with methylene blue.
Figure 3. The specificity of 5-Hydroxymethylcytosine (5-hmC) (HMC31) Mouse mAb #51660 was determined by an ELISA. The antibody was titrated against a single-stranded DNA oligo containing either unmodified cytosine or differentially modified cytosine (5-mC, 5-hmC, 5-caC, or 5-fC). As shown in the graph, the antibody only binds to the oligo containing 5-hmC.
Figure 4. The specificity of 5-Hydroxymethylcytosine (5-hmC) (HMC31) Mouse mAb #51660 was determined by MeDIP. DNA IPs were performed with genomic DNA prepared from mouse embryonic stem cells, spiked with DNA containing either unmodified cytosine, 5-methylcytosine (5-mC), or 5-hydroxymethylcytosine (5-hmC). IPs were performed using SimpleDIP™ Hydroxymethylated DNA IP (hMeDIP) Kit #95176. The enriched DNA was quantified by real-time PCR using primers specific to the spiked-in control DNA sequence. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input DNA, which is equivalent to one.
|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 (20mg/ml)||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|
|31482||SimpleDIP™ Cell Lysis Buffer|
|49291||SimpleDIP™ DNA IP Buffer (10X)|
|7009||ChIP Elution Buffer (2X)|
|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|
|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|
|7017 / 14654||Magnetic Separation Rack|
|9872||Phosphate Buffered Saline (PBS-1X) pH7.2 (Sterile)|
|-||Phenol/Choloroform/Isoamyl Alcohol (25:24:1) Saturated with 10 mM Tris, pH 8.0, 1 mM EDTA|
|-||Chloroform:Isoamyl Alcohol (24:1)|
|-||Taq DNA polymerase|
|-||Real-Time PCR SYBR™ Green Reaction Mix|
|Number of Cells Used||Approximate Yield|
|1 million||6 µg|
|5 million||30 µg|
|10 million||60 µg|
For suspension cells, count cells using a hemocytometer.
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.
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.
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.
|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|
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.
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.
|Primer length||24 nucleotides|
|Amplicon size||150 to 200 bp (for standard PCR) 80 to 160 bp (for real-time quantitative PCR)|
|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|
|a.||Initial Denaturation||95°C||5 min|
|e.||Repeat Steps b-d for a total of 34 cycles.|
|f.||Final Extension||72°C||5 min|
|Reagent||Volume for 1 PCR Reaction (18 µl)|
|Nuclease-free H2O||6 µl|
|5 µM primers||2 µl|
|SYBR™ Green Reaction Mix||10 µl|
|a.||Initial Denaturation||95°C||3 min|
|d.||Repeat step b and c for a total of 40 cycles|
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
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.
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.
|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
Protocol Id: 844
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.
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.
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SimpleDIP is a trademark of Cell Signaling Technology, Inc.
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