5-Hydroxymethylcytosine (5-hmC) (HMC31) Mouse mAb #51660

SimpleDIP^™ Hydroxymethylated DNA IP (hMeDIP) Protocol

Required Reagents

Reagents Included

Reagents Not Supplied

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.

1. 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.

2. 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.
3. Resuspend the cell pellet from step 2 in 500 µl of SimpleDIP^™ Cell Lysis Buffer.
4. 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.
5. Add 500 µl of phenol/chloroform/isoamyl alcohol (25:24:1) and mix thoroughly by vortexing for 30 sec.
6. 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.
7. Add 500 µl of chloroform/isoamyl alcohol (24:1) to the material and mix thoroughly by vortexing for 30 sec.
8. 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.
9. 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.
10. Pellet DNA by centrifugation at 10,000 x g for 5 min in a microcentrifuge.
11. Carefully remove supernatant and wash pellet with 70% ethanol chilled at -20°C. Decant supernatant and air dry or vacuum dry pellet.
12. Resuspend pellet in 500 µl of TE Buffer and add 2 µl of RNase A. Incubate for 30 min at 37°C.
13. 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.

2. Remove a 50 µl sample of the diluted DNA and transfer to a microfuge tube. This is the 10% input sample.

3. 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.

4. 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.
5. 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:

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.

4. Start the following PCR reaction program:

5. 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.

4. Start the following PCR Reaction program:

5. 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 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