ChIP Protocol (Native & Cross-Linking)

Native ChIP Protocol

Native ChIP is a technique used for studying naturally occurring protein-DNA interactions. It is usually specific to histone modifications and native chromatin is used as the starting chromatin. Micrococcal nuclease first cuts the chromatin linker DNA, which yields fragments of intact nucleosomes from 200bp-1000bp. The DNA fractions of interest can be pulled down by the specific antibody-antigen reactions. Therefore, this process enriches for the DNA fragments bound by target proteins. Finally, DNA can be purified from the complex and analyzed with PCR or qPCR protocols.

Native ChIP diagram courtesy of He C. and Bonasio R. Click to enlarge.

Solution and reagents:

Sample preparation:

Use cell culture from 10cm culture dishes with 20mL of medium. Cells are ready to be used for a ChIP assay when the density of cells reaches 80%-90%. The abundance of target protein depends upon the protein of interest and the number of cells. Refer to the table below:

For optimal results, we recommend a starting amount of >4×10⁶ cells for histone protein and RNA polymerase II. Transcription factors or cofactors may require more cells.

1. Take out the culture dishes and discard the culture medium.
2. Wash the cells with ice-cold PBS three times.
3. Add 1ml of PBS and collect the cells by scraping the cells from culture dishes.
4. Centrifuge for 3min, 4 ℃ at 2500 RPM.
5. Resuspend the cells by adding reaction buffer with fresh protease inhibitors (500μl for every 2x10⁷ cells) to lyse the cells on ice for 10 min.
6. Centrifuge for 3min, 4 ℃ at 2500 RPM.
7. Resuspend the pellet by adding 1mL reaction buffer and micrococcal nuclease to react at 37 ℃ for 20 min. Refer to the instructions of the micrococcal nuclease to determine the enzymolysis condition. Determine the optimal processing time and concentration of micrococcal nuclease with a previous preliminary experiment.
8. Add 5mM EDTA to terminate the enzymatic hydrolysis

Sonication

Sonication conditions may differ depending on the sonicator and type of cells used. We recommend using the EpiSonic 2000 Sonication System. Depending on the different cell lines, the ideal fragment size of DNA after sonication is 200-1000bp. See our Sonication Application protocol to determine the ideal size for your experiment.

1. Sonicate to obtain the ideal fragment size of DNA (200-1000bp). Sonication conditions may differ depending on the sonicator or type of cells used.
2. Centrifuge for 2min, 4℃ at 2500 RPM. Transfer the supernatant into a new tube.
3. To verify sonication results, 5μl of sheared chromatin supernatant can be used for agarose gel analysis/Bioanalyzer.

Immunoprecipitation:

1. Transfer 500μl of supernatant (containing DNA from 1x10⁷ cells).
2. Prepare four groups of sample conditions :
   

   Experimental group: add 2-5μg of specific antibody into 500μl supernatant and place tube on a rotary mixer at 4℃ overnight to form the antigen-antibody complex.
   Input group: follow the same procedure as the experimental group. However, do not add antibodies.
   Negative control: follow the same procedure as the experimental group, but with a normal rabbit IgG antibody control.
   Positive control: follow the same procedure as the experimental group but with a histone H3 or RNA polymerase II antibody control.

3. Add 50μl of magnetic beads to the complex solution and incubate at 4 ^oC for 2 to 4 hours.
4. Place tube on magnetic stand and remove the supernatant.

Wash the beads:

Wash the beads with filtered tips:

a. 2×1ml RIPA buffer
b. 2×1ml RIPA buffer +0.3M NaCl
c. 2×1ml LiCl buffer
d. 2× 1ml TE buffer + 0.2%Triton X-100
e. 1× 1ml TE buffer. Mix 1 min on rotator.

Wash the beads two times with the buffers above. Pipette 3-8 times by tips and rotate for 10 min each.

1. Remove the wash buffer by placing tubes on a magnetic stand.
2. Re-suspend the precipitation complex in 100μl TE buffer. Add 3-5ul of 10% SDS and 5ul of 20mg/ml proteinase K. Incubate at 65 ℃ overnight.
3. The next day, vortex briefly. Place sample tubes on magnetic stand and transfer supernatant to a new tube .
4. Wash beads with 100μl TE buffer + 0.5M NaCl. Combine with the supernatant in step 3.

DNA purification:

DNA can be extracted by using your preferred method and procedure. We recommend using the EpiNext DNA Purification HT System and following the procedure found in the user guide.

Detection:

Perform a PCR or Real time PCR assay to analyze the DNA associated with the target protein.

Cross-linking ChIP Protocol

ChIP is an immunoprecipitation technique used to study interactions between proteins and DNA in a cell. The goal is to determine what specific proteins are associated with different regions in the genome. The target protein is cross-linked together with DNA, and then the DNA is broken into smaller fragments with sonication or enzymatic hydrolysis. The DNA fractions of interest can be pulled down by specific antibody-antigen reactions. Therefore, this process enriches for the DNA fragments bound by target proteins. Finally, DNA can be purified from the complex and analyzed with PCR or qPCR protocols.

X ChIP diagram courtesy of He C. and Bonasio R. Click to enlarge.

Solution and reagents:

Sample preparation:

Cross-linking and lysis - for transcription factor and cofactors

Use cell culture from 10cm culture dishes with 20mL of culture medium. Cells are ready to be used for a ChIP assay when the density of cells reaches 80%-90%. The abundance of target protein depends upon the protein of interest and the number of cells. Refer to the table below:

For optimal results, a starting amount of >4×10⁶ cells for histone protein and RNA polymerase II is recommended. Transcription factors and cofactors may require more cells. 1% formaldehyde is used to crosslink proteins and DNA. This process is time-dependent and needs to be optimized for different types of cells. Crosslinking deficiency may result in a false negative. Excessive crosslinking may cause masking or changing of epitopes and protein-protein cross-linking may yield false positives. Therefore, crosslinking the sample for 10min is recommended. The crosslinking reaction can be reversed by glycine.

1. To prepare a 1% formaldehyde solution, take out the culture dishes and add 550μl of 37% formaldehyde into the 20ml of culture medium. Mix the medium.
2. Place the culture dishes at RT for 10 min.
3. Add 125mM glycine solution to terminate the cross-link reaction. Gently mix the solution.
4. Incubate the culture dishes at RT for 5 min.
5. Remove culture medium and wash the cells with ice-cold PBS three times.
6. Add 1mL of ice-cold PBS into the dishes and scrape the cells from the dishes quickly.
7. Wash the bottom of dishes with PBS for 2 times with proper volume. Aspirate the PBS into the tube from step 6.
8. Centrifuge for 3min, 4℃ at 2500 RPM to collect the pellets.
9. Add the appropriate volume of ChIP lysis buffer with fresh protease inhibitors(1ml for every 2×10⁷ cells) to suspend the cells. Next, lyse the cells on ice for 15 min

Sonication

Sonication conditions may differ depending on the sonicator and type of cells used. We recommend using the EpiSonic 2000 Sonication System. Depending on the different cell lines, the ideal fragment size of DNA after sonication is 200-1000bp. See our Sonication Application protocol to determine the ideal size for your experiment.

1. Sonicate to obtain the ideal fragment size of DNA (200-1000bp). Sonication conditions may differ depending on the sonicator or type of cells used.
2. Centrifuge for 10min, 4℃ at 12000 RPM. Transfer the supernatant into a new tube.
3.  Use 50μl of supernatant for an agarose gel analysis to validate the effect of sonication.

Determination of DNA fragment size

1. Add 70μl elution buffer to the 50μl of chromatin.
2. Add 4.8μl 5M NaCl and 2μl of 10mg/ml RNase A, then incubate at 65℃ overnight. The purpose is to remove interference from RNA in the sample.
3. The next day, add 2μl of 20mg/ml proteinase K and incubate at 60℃ for 1 hour. The aim is to break the reactions between protein and DNA so that the DNA can be purified.
4. Use a DNA purification kit to enrich the DNA (or perform a phenol-chloroform extraction and ethanol precipitation method).
5. A 2% agarose gel can be prepared to detect the efficiency of sonication.

Immunoprecipitation:

1. Transfer 500μl of chromatin (diluted by RIPA buffer) containing DNA from 1×10⁷ cells into a new tube.
2. Prepare four groups of sample conditions :
   

   Experimental group: add 2-5μg of specific antibody into 500μl cell lysate and place tube on a rotary mixer at 4℃ overnight to form the antigen-antibody complex.
   Input group: follow the same procedure as the experimental group. However, do not add antibodies.
   Negative control: follow the same procedure as the experimental group, but with a normal rabbit IgG antibody control.
   Positive control: follow the same procedure as the experimental group but with a histone H3 or RNA polymerase II antibody control.

3. Add 50μl of magnetic beads to the complex solution and incubate at 4 ^oC for 2 to 4 hours.
4. Place tube on magnetic stand and remove the supernatant.

Wash the beads

Wash the beads with filtered tips:

1. 2×1ml Low salt wash buffer
2. 2×1ml High salt wash buffer
3. 2×1ml LiCl buffer
4. 2×1ml TE buffer

Wash the beads two times with the buffers above. Pipette 3-8 times by tips and rotate for 10 min each. Remove wash buffer by placing tubes on magnetic stand.

Reverse crosslinking

1. Add 120μl Elution buffer to the precipitation complex and gently mix. Place tubes on rotating mixer at RT for 15 min. Repeat this step once more for a total of two times.
2. Place tubes on magnetic stand and transfer the supernatant to a new tube.
3. Add 9.6μl of 5M NaCl and 2μl of 10mg/ml RNase into all sample groups. Incubate at 65℃ overnight to reverse crosslinkage.
4. The next day, add 2μl of 20mg/ml proteinase K and incubate at 60℃ for 1 hour.

DNA purification

DNA can be extracted by using your preferred method and procedure. We recommend using the EpiNext DNA Purification HT System and following the procedure found in the user guide.

Detection

Perform a PCR or Real-time PCR assay to analyze the DNA associated with the target protein.