Required Reagents

Reagents Included in SimpleChIP® Enzymatic Chromatin IP Kit (Agarose Beads) #9002:

  1. Glycine Solution (10X)
  2. Buffer A (4X)
  3. Buffer B (4X)
  4. ChIP Buffer (10X)
  5. ChIP Elution Buffer (2X)
  6. 5 M NaCl
  7. 0.5 M EDTA
  8. 1M DTT
  9. DNA Binding Reagent A
  10. DNA Wash Reagent B
  11. DNA Elution Reagent C
  12. DNA Spin Columns
  13. Protease Inhibitor Cocktail (200X)
  14. RNAse A (10 mg/ml)
  15. Micrococcal Nuclease (2000 gel units/µl): NEB #M0247S
  16. Proteinase K (20 mg/ml): NEB #P8102S
  17. SimpleChIP® Human RPL30 Exon 3 Primers #7014
  18. SimpleChIP® Mouse RPL30 Intron 2 Primers #7015
  19. Histone H3 (D2B12) XP® Rabbit mAb (ChIP Formulated) #4620
  20. Normal Rabbit IgG #2729
  21. ChIP-Grade Protein G Agarose Beads #9007

Reagents Not included in SimpleChIP® Enzymatic Chromatin IP Kit (Agarose Beads) #9002:

  1. PMSF (0.1 M in ethanol)
  2. Formaldehyde (37%)
  3. Ethanol (96-100%)
  4. Isopropanol
  5. 1X PBS
  6. Nuclease-free water
  7. Taq DNA polymerase
  8. dNTP Mix

A. In Vivo Crosslinking, Nuclei Preparation, and Nuclease S7 Digestion of Chromatin

Before starting:
Stimulate or treat approximately 4 X 107 cells for each experiment. This number of cells will generate one chromatin preparation that can be used for up to 10 separate immunoprecipitations.

  • For HeLa cells, this is equivalent to 5 x 15 cm culture dishes containing cells that are 90% confluent in 20 ml of growth media.
  • Include one extra dish to be used for determination of cell number using a hemocytometer.

Remove and warm:

  • 200X Protease Inhibitor Cocktail
  • 1 M DTT
  • 0.1 M PMSF (not supplied)
  • 10X ChIP Buffer and ensure SDS is completely in solution.

Prepare and chill on ice for each set of 4 X 107 cells (5 dishes):

  • 10 ml 10X glycine
  • 200 ml 1X PBS (not supplied)
  • 10 ml 1X PBS + 100 µl PMSF
  • 10 ml 1X Buffer A (2.5 ml 4X Buffer A + 7.5 ml water) + 5µl 1 M DTT + 50 µl 200X Protease Inhibitor Cocktail (PIC) + 100 µl PMSF
  • 11 ml 1X Buffer B (2.75 ml 4X Buffer B + 8.25 ml water) + 5.5 µl 1 M DTT
  • 1 ml 1X ChIP Buffer (100 µl 10X ChIP Buffer + 900 µl water) + 5 µl 200X Protease Inhibitor Cocktail (PIC) + 10 µl PMSF.
  1. To crosslink proteins to DNA, add 540 µl of 37% formaldehyde to each 15 cm culture dish containing 20 ml medium. Swirl briefly to mix and incubate for 10 minutes at room temperature.
    • Final formaldehyde concentration is 1%.
    • Use fresh formaldehyde that is not past the manufacturer’s expiration date.
    • Addition of formaldehyde may result in a color change of the medium.
  2. Add 2 ml of 10X glycine to each 15 cm dish, swirl briefly to mix, and incubate for 5 minutes at room temperature.
    • Addition of glycine may result in a color change of the medium.
  3. For suspension cells, transfer cells to a 50 ml conical tube, centrifuge at 1,500 rpm in a bench top centrifuge for 5 minutes at 4°C, and wash pellet two times with 20 ml ice-cold 1X PBS. Immediately proceed to step 5.
  4. For adherent cells, remove media and wash cells two times with 20 ml ice-cold 1X PBS, completely removing wash from culture dish each time. Add 2 ml ice-cold 1X PBS + PMSF to the 15 cm dish of cells. Scrape and resuspend cells into cold buffer. Combine cells from all 5 plates into one 15 ml conical tube. Centrifuge cells at 1,500 rpm in a bench top centrifuge for 5 minutes at 4°C.
  5. Remove supernatant and resuspend cells in 10 ml ice-cold Buffer A + DTT + PIC + PMSF. Incubate on ice for 10 minutes. Mix by inverting tube every 3 minutes.
  6. Pellet nuclei by centrifugation at 3,000 rpm in a bench top centrifuge for 5 minutes at 4°C. Remove supernatant and resuspend pellet in 10 ml ice-cold Buffer B + DTT. Repeat centrifugation, remove supernatant, and resuspend pellet in 1.0 ml Buffer B + DTT. Transfer sample to a 1.5 ml microcentrifuge tube.
  7. Add 5 µl of Micrococcal Nuclease, mix by inverting tube several times and incubate for 20 minutes at 37°C with frequent mixing to digest DNA to length of approximately 150-900 bp. Mix by inversion every 3 to 5 minutes.
    • The amount of Micrococcal Nuclease required to digest DNA to the optimal length may need to be determined empirically for individual cell lines (see Appendix A).
    • HeLa nuclei digested with 5 µl Micrococcal Nuclease per 4 X 107 cells in 1 ml Buffer B + DTT gave the appropriate length DNA fragments.
  8. Stop digest by adding 100 µl of 0.5 M EDTA and placing tube on ice.
  9. Pellet nuclei by centrifugation at 13,000 rpm in a microcentrifuge for 1 minute at 4°C and remove supernatant.
  10. Resuspend nuclear pellet in 1 ml of 1X ChIP buffer + PIC + PMSF and split into two tubes of 500 µl. Incubate on ice for 10 minutes.
  11. Sonicate each tube of lysate with several pulses to break nuclear membrane. Incubate samples for 30 seconds on wet ice between pulses.
    • Optimal conditions required for complete lysis of nuclei can be determined by observing nuclei under light microscope before and after sonication.
    • HeLa nuclei were completely lysed after 3 sets of 20-second pulses using a VirTis Virsonic 100 Ultrasonic Homogenizer/Sonicator at setting 6 with a 1/8-inch probe.
    • Alternatively, nuclei can be lysed by homogenizing the lysate 20 times in a Dounce homogenizer; however, lysis may not be as complete.
  12. Clarify lysates by centrifugation at 10,000 rpm in a microcentrifuge for 10 minutes at 4°C.
  13. Transfer supernatant to a new tube. This is the cross-linked chromatin preparation, which should be stored at -80°C until further use. Remove 50 µl of the chromatin preparation for Analysis of Chromatin Digestion and Concentration (Section B).

B. Analysis of Chromatin Digestion and Concentration (Recommended Step)

  1. To the 50 µl chromatin sample (from Step 13 in Section A), add 100 µl nuclease-free water, 6 µl 5 M NaCl and 2 µl RNAse A. Vortex to mix and incubate samples at 37°C for 30 minutes.
  2. To each RNAse A-digested sample, add 2 µl Proteinase K. Vortex to mix and incubate samples at 65°C for 2 hours.
  3. Purify DNA from samples using spin columns as described in Section F.
  4. After purification of DNA, remove a 10 µl sample and determine DNA fragment size by electrophoresis on a 1% agarose gel with a 100 bp DNA marker. DNA should be digested to a length of approximately 150-900 bp (1 to 5 nucleosomes).
  5. To determine DNA concentration, transfer 2 µl of purified DNA to 98 µl TE 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 200 µg/ml.

NOTE: For optimal ChIP results, it is highly critical that the chromatin is of appropriate size and concentration. Over-digestion of chromatin may diminish signal in the PCR quantification. Under-digestion of chromatin may lead to increased background signal and lower resolution. Adding too little chromatin to the IP may result in diminished signal in the PCR quantification. A protocol for optimization of chromatin digestion can be found in Appendix A.

C. Chromatin Immunoprecipitation

Before starting:
Remove and warm:

  • 200X Protease Inhibitor Cocktail
  • 10X ChIP Buffer and ensure SDS is completely in solution.

Remove and place on ice:

NOTE: The cross-linked chromatin preparation does not need to be diluted as described below. Antibodies can be added directly to the undiluted chromatin preparation for immunoprecipitation of chromatin complexes.

  1. In one tube, prepare enough 1X ChIP Buffer for the desired number of immunoprecipitations. Each precipitation contains 400 µl of 1X ChIP Buffer (40 µl of 10X ChIP Buffer + 360 µl water) and 2 µl Protease Inhibitor Cocktail. When determining the number of immunoprecipitations, the user should include the positive control Histone H3 (D2B12) XP® Rabbit mAb (ChIP Formulated) #4620 and negative control Normal Rabbit IgG #2729 samples. Place mix on ice.
  2. To the prepared ChIP buffer, add the equivalent of 100 µl (5 to 10 µg of chromatin DNA) of the cross-linked chromatin preparation (from Step 13 in Section A) for each immunoprecipitation. For example, for 10 immunoprecipitations, prepare a tube containing 4 ml 1X ChIP Buffer (400 µl 10X ChIP Buffer + 3.6 ml water) + 20 µl Protease Inhibitor Cocktail + 1 ml digested chromatin preparation.
  3. Remove a 10 µl sample of the diluted chromatin and transfer to a microfuge tube. This is your 2% Input Sample, which can be stored at -20°C until further use (Step 1 in Section E).
  4. For each immunoprecipitation, transfer 500 µl of the diluted chromatin to a microcentrifuge tube and add the immunoprecipitating antibody. The amount of antibody required for IP varies for each antibody and should be determined by the user. For the positive control Histone H3 (D2B12) XP® Rabbit mAb (ChIP Formulated) #4620, add 10 µl to the IP sample. For the negative control Normal Rabbit IgG #2729, add 1 µl (1 µg) to 5 µl (5 µg) to the IP sample. Incubate IP samples 4 hours to overnight at 4°C with rotation.
  5. Add 30 µl of ChIP-Grade Protein G Agarose Beads #9007 and incubate for 2 hours at 4°C with rotation.
  6. Proceed to Section D.

D. Washing of the Immunoprecipitated Chromatin

Before starting:
Remove and warm:

  • 10X ChIP Buffer and ensure SDS is completely in solution.

Prepare and chill on ice for each IP:

  • Low salt wash: 3 ml 1X ChIP Buffer (300 µl 10X ChIP Buffer + 2.7 ml water)
  • High salt wash: 1 ml 1X ChIP Buffer (100 µl 10X ChIP Buffer + 900 µl water) + 70 µl 5 M NaCl
  1. Pellet Protein G Agarose Beads in each immunoprecipitation (from Step 5, Section C) by brief 1 minute centrifugation at 6,000 rpm and remove supernatant.
  2. Add 1 ml of low salt wash to the beads and incubate at 4°C for 5 minutes with rotation. Repeat steps 1 and 2 two additional times, for a total of 3 low salt washes.
  3. Add 1 ml of high salt wash to the beads and incubate at 4°C for 5 minutes with rotation. Immediately proceed to Section E.

E. Elution of Chromatin from Antibody/Protein G Beads and Reversal of Cross-links

Before starting:

  • Remove and warm 2X ChIP Elution Buffer in a 37°C water bath and ensure SDS is in solution.
  • Set a water bath or thermomixer to 65°C.
  • Prepare 150 µl 1X ChIP Elution Buffer (75 µl 2X ChIP Elution Buffer + 75 µl water) for each washed IP (from Step 3 in Section D) and the 2% input sample (from Step 3 in Section C).
  1. Add 150 µl of the 1X ChIP Elution Buffer to the 2% input sample tube and set aside at room temperature until Step 7.
  2. Pellet Protein G Agarose Beads by brief 1 minute centrifugation at 6,000 rpm and remove supernatant.
  3. Add 150 µl 1X ChIP Elution Buffer to each IP sample.
  4. Elute chromatin from the antibody/Protein G beads for 30 minutes 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.
  5. Pellet Protein G Agarose Beads by brief 1 minute centrifugation at 6,000 rpm.
  6. Carefully transfer eluted chromatin supernatant to a new tube.
  7. To all tubes, including the 2% input sample from Step 1, add 6 µl 5 M NaCl and 2 µl Proteinase K and incubate 2 hours at 65°C.
  8. Immediately proceed to Section F. Alternatively, samples can be stored at -20°. However, to avoid formation of a precipitate, be sure to warm samples to room temperature before adding DNA Binding Reagent A (Section F, Step 1).

F. DNA Purification Using Spin Columns

Before starting:

  • Add 12 ml of isopropanol to DNA Binding Reagent A and 24 ml of ethanol (96-100%) to DNA Wash Reagent B before use. These steps only have to be performed once prior to the first set of DNA purifications.
  • Remove one DNA purification spin column and collection tube for each DNA sample from Section E.
  1. Add 600 μl DNA Binding Reagent A to each DNA sample and vortex briefly.
    • 4 volumes of DNA Binding Reagent A should be used for every 1 volume of sample.
  2. Transfer 375 μl of each sample from Step 1 to a DNA purification spin column in collection tube.
  3. Centrifuge at 14,000 rpm 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 375 μl of each sample from Step 1 to the spin column in collection tube. Repeat Steps 3 and 4.
  6. Add 700 μl of DNA Wash Reagent B to the spin column in collection tube.
  7. Centrifuge at 14,000 rpm in a microcentrifuge for 30 sec.
  8. Remove the spin column from the collection tube and discard the liquid. Replace spin column in the collection tube.
  9. Centrifuge at 14,000 rpm in a microcentrifuge for 30 sec.
  10. Discard collection tube and liquid. Retain spin column.
  11. Add 50 μl of DNA Elution Reagent C to each spin column and place into a clean 1.5 ml microcentrifuge tube.
  12. Centrifuge at 14,000 rpm in a microcentrifuge for 30 sec to elute DNA.
  13. Remove and discard DNA purification spin column. Eluate is now purified DNA. Samples can be stored at -20°C.

G. 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 human or mouse RPL30 gene and can be used for either standard PCR or quantitative real-time PCR. If the user is performing ChIPs from another species, it is recommended that the user design the appropriate specific primers to DNA and determine the optimal PCR conditions.
  • 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 2% input sample, the positive control Histone H3 sample, the negative control Normal Rabbit IgG sample, 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 H2O 12.5 µl
10X PCR Buffer 2.0 µl
4 mM dNTP Mix 1.0 µl
5 µM RPL30 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% poly-acrylamide gel electrophoresis with a 100 bp DNA marker. The expected size of the PCR product is 161 bp for human RPL30 and 159 bp for mouse RPL30.

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 positive control Histone H3 sample, the negative control Normal Rabbit IgG sample, a tube with no DNA to control for contamination, and a serial dilution of the 2% input chromatin 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 RPL30 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 and Extension: 60°C 60 sec
d. Repeat steps 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 = 2% x 2(C[T] 2%Input Sample – C[T] IP Sample)

C[T] = CT = Threshold cycle of PCR reaction

Appendix A: Optimization of Chromatin Digestion

Optimal conditions for digestion of cross-linked DNA to 150-900 base pairs in length depend on cell type and cell concentration as well as the concentration of Micrococcal Nuclease. Below is a protocol to determine the optimal digestion conditions for a specific cell type and concentration of cells.

  1. Prepare cross-linked nuclei from 4 X 107 cells as described in Section A, Steps 1-6.
  2. Transfer 200 µl of the nuclei preparation from Step 6 in Section A into 5 individual microcentrifuge tubes and place on ice.
  3. Add 5 µl Micrococcal Nuclease stock to 20 µl of 1X Buffer B + DTT (1:5 dilution of enzyme).
  4. To each of the 5 tubes in Step 2, add 0 µl, 2.5 µl, 5 µl, 7.5 µl or 10 µl of the diluted Micrococcal Nuclease, mix by inverting tube several times and incubate for 20 min at 37°C with frequent mixing.
  5. Stop digest by adding 20 µl of 0.5 M EDTA and placing tubes on ice.
  6. Pellet nuclei by centrifugation at 13,000 rpm in a microcentrifuge for 1 min at 4°C and remove supernatant.
  7. Resuspend nuclear pellet in 200 µl of 1X ChIP buffer + PIC +PMSF. Incubate on ice for 10 min.
  8. Sonicate lysate with several pulses to break nuclear membrane. Incubate samples 30 sec on wet ice between pulses.
    • Optimal conditions required for complete lysis of nuclei can be determined by observing nuclei under light microscope before and after sonication.
    • HeLa nuclei were completely lysed after 3 sets of 20-second pulses using a VirTis Virsonic 100 Ultrasonic Homogenizer/Sonicator set at setting 6 with a 1/8-inch probe.
    • Alternatively, nuclei can be lysed by homogenizing the lysate 20 times in a Dounce homogenizer; however, lysis may not be as complete.
  9. Clarify lysates by centrifugation at 10,000 rpm in a microcentrifuge for 10 min at 4°C.
  10. Transfer 50 µl of each of the sonicated lysates to new microfuge tubes.
  11. To each 50 µl sample, add 100 µl nuclease-free water, 6 µl 5 M NaCl and 2 µl RNAse A. Vortex to mix and incubate samples at 37°C for 30 min.
  12. To each RNAse A-digested sample, add 2 µl Proteinase K. Vortex to mix and incubate sample at 65°C for 2 hours.
  13. Remove 20 µl of each sample and determine DNA fragment size by electrophoresis on a 1% agarose gel with a 1 kb DNA marker.
  14. Observe which of the digestion conditions produces DNA in the desired range of 150-900 base pairs (1 to 5 nucleosomes). The volume of diluted Micrococcal Nuclease that produces the desired size of DNA fragments using this optimization protocol is equivalent to the volume of Micrococcal Nuclease stock that will need to be added to 4 X 107 cells to produce the desired size of DNA fragments. For example, if 5 µl of diluted Micrococcal Nuclease produces DNA fragments of 150-900 base pairs in this protocol, then 5 µl of stock Micrococcal Nuclease should be added to 4 X 107 cells during the digestion of chromatin in Step 7 of Section A.
  15. If results indicate that DNA is not in the desired size range, then repeat optimization protocol, adjusting the amount of Micrococcal Nuclease in each digest accordingly.

Appendix B: Troubleshooting Guide

Problem Possible Causes Recommendation
1. Concentration of the digested chromatin is too low. Not enough cells added to the chromatin digestion or nuclei were not completely lysed after digestion. If DNA concentration of the chromatin preparation is close to 50 µg/ml, add additional chromatin to each IP to give 5 µg/IP and continue with protocol.
Count a separate plate of cells before cross-linking to determine an accurate cell number and/or visualize nuclei under microscope before and after sonication to confirm complete lysis of nuclei.
2. Chromatin is under-digested and fragments are too large (greater than 900 bp). Cells may have been over cross-linked. Cross-linking for longer than 10 min may inhibit digestion of chromatin. Shorten the time of cross-linking to 10 min.
Too many cells or not enough Micrococcal Nuclease was added to the chromatin digestion. Count a separate plate of cells before cross-linking to determine accurate cell number and see Appendix A for optimization of chromatin digestion.
3. Chromatin is over-digested and fragments are too small (exclusively 150 bp mono-nucleosome length). Complete digestion of chromatin to mono-nucleosome length DNA may diminish signal during PCR quantification, especially for amplicons greater than 150 bp in length. Not enough cells or too much Micrococcal Nuclease added to the chromatin digestion. Count a separate plate of cells before cross-linking to determine accurate cell number and see Appendix A for optimization of chromatin digestion.
4. No product or very little product in the input PCR reactions. Not enough DNA added to the PCR reaction or conditions are not optimal. Add more DNA to the PCR reaction or increase the number of amplification cycles.
PCR amplified region may span nucleosome-free region. Optimize the PCR conditions for experimental primer set using purified DNA from cross-linked and digested chromatin. Design a different primer set and decrease length of amplicon to less than 150 bp (see primer design recommendations in Section G).
Not enough chromatin added to the IP or chromatin is over-digested. For optimal ChIP results, add 5 to 10 µg chromatin per IP. See recommendations for problems 1 and 3 above.
5. No product in the positive control Histone H3-IP RPL30 PCR reaction. Not enough chromatin or antibody added to the IP reaction or IP incubation time is too short. Be sure to add 5 to 10 µg of chromatin and 10 µl of antibody to each IP reaction and incubate with antibody over-night and an additional 2 h after adding Protein G beads.
Incomplete elution of chromatin from Protein G beads. Elution of chromatin from Protein G beads is optimal at 65°C with frequent mixing to keep beads suspended in solution.
6. Quantity of product in the negative control Rabbit IgG-IP and positive control Histone H3-IP PCR reactions is equivalent. Too much chromatin or antibody added to the IP reaction. Add no more than 15 µg of chromatin and 10 µl of Histone H3 antibody to each IP reaction. Reduce the amount of normal rabbit IgG to 1 µl per IP.
Too much DNA added to the PCR reaction or too many cycles of amplification. Add less DNA to the PCR reaction or decrease the number of PCR cycles. It is very important that the PCR products are analyzed within the linear amplification phase of PCR. Otherwise, the differences in quantities of starting DNA can not be accurately measured.
7. No product in the Experimental Antibody-IP PCR reaction. Not enough DNA added to the PCR reaction. Add more DNA to the PCR reaction or increase the number of amplification cycles.
Not enough antibody added to the IP reaction. Typically a range of 1 to 5 µg of antibody are added to the IP reaction; however, the exact amount depends greatly on the individual antibody. Increase the amount of antibody added to the IP.
Antibody does not work for IP. Find alternative antibody source.

posted March 2008

revised April 2010

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