SimpleChIP® Chromatin Immunoprecipitation Protocol (Magnetic Beads)

Specific for product: SimpleChIP^® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003.

I. Cell Culture Cross-linking and Sample Preparation:

For optimal ChIP results, use approximately 4 X 10⁶ cells for each immunoprecipitation to be performed (at least 12 X 10⁶ cells are required in order to include positive and negative controls). For HeLa cells, one IP is equivalent to half of a 15 cm culture dish containing cells that are 90% confluent in 20 ml of growth medium. One additional sample should be processed for Analysis of Chromatin Digestion and Concentration (Section III). Since every cell type is different, we recommend including one extra dish of cells for each cell line used in the experiment to be used for determination of cell number using a hemocytometer or cell counter.

Before starting:

(!) All buffer volumes should be increased proportionally based on the number of 15 cm tissue culture dishes (each containing 20 ml medium) for adherent cells or per 20 ml of suspension cells used in the experiment.

• Warm Protease Inhibitor Cocktail (200X) #7012 (PIC) and Glycine Solution (10X) #7005. Make sure PIC is completely thawed.
• Prepare 2 ml of Phosphate Buffered Saline (PBS) #9872 + 10 µl 200X PIC #7012 per 15 cm tissue culture dish (containing 20 ml medium) for adherent cells or 20 ml of suspension cells to be processed and place on ice.
• Prepare 40 ml of PBS per 15 cm tissue culture dish (containing 20 ml medium) for adherent cells or 20 ml of suspension cells to be processed and place on ice.
• Prepare 540 µl of 37% formaldehyde or 1,250 µl of 16% Formaldehyde, Methanol-Free #12606 per 15 cm tissue culture dish (containing 20 ml medium) for adherent cells or 20 ml of suspension cells to be processed and keep at room temperature. Use fresh formaldehyde that is not past the manufacturer's expiration date.

1. To crosslink proteins to DNA, add formaldehyde to a final concentration of 1%. For adherent cells, add 540 µl of 37% formaldehyde or 1,250 µl of 16% Formaldehyde, Methanol-Free #12606 to each 15 cm dish containing 20 ml medium. For suspension cells, ensure the density is below 0.5 x 10⁶ cells/ml and add the same volume of formaldehyde as described above to 20 ml medium. Swirl briefly to mix and incubate for 10 minutes at room temperature.

   NOTE: Addition of formaldehyde may result in a color change of the medium.

2. Add 2 ml of Glycine Solution (10X) #7005 to each 15 cm tissue culture dish (containing 20 ml medium) for adherent cells or 20 ml of suspension cells, swirl briefly to mix, and incubate for 5 min at room temperature.

   NOTE: 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 2,000 x g in a benchtop centrifuge 5 min at 4°C and wash pellet two times with 20 ml ice-cold PBS. Remove supernatant and immediately continue with Nuclei Preparation and Chromatin Digestion (Section II). (SAFE STOP) Alternatively samples may be stored at -80°C for up to 3 months.
4. For adherent cells:
   1. Remove media and wash cells two times with 20 ml ice-cold 1X PBS, completely removing wash from culture dish each time.
   2. Add 2 ml ice-cold PBS + PIC to each 15 cm dish. Scrape cells into the cold buffer. Combine cells from all culture dishes into one 15 ml conical tube.
   3. Centrifuge cells at 2,000 x g in a benchtop centrifuge for 5 min at 4°C. Remove supernatant and immediately continue with Nuclei Preparation and Chromatin Digestion (Section II). (SAFE STOP) Alternatively samples may be stored at -80°C for up to 3 months.

II. Nuclei Preparation and Chromatin Digestion

Before starting:

(!) All buffer volumes should be increased proportionally based on the number of IP preps (4 x 10⁶ tissue culture cells) in the experiment.

• Warm Protease Inhibitor Cocktail (200X) #7012 (PIC). Make sure it is completely thawed prior to use.
• (!!) Prepare 1 M DTT (192.8 mg DTT #7016 + 1.12ml dH₂O). Make sure DTT crystals are completely in solution. Once in solution, store 1M DTT at -20°C.
• Warm 10X ChIP Buffer #7008 and ensure SDS is completely in solution.
• Prepare 1 ml 1X Buffer A (250 µl 4X Buffer A #7006 + 750 µl water) + 0.5 µl 1M DTT + 5 µl 200X PIC #7012 per IP prep and place on ice.
• Prepare 1.1 ml 1X Buffer B (275 µl 4X Buffer B #7007 + 825 µl water) + 0.55 µl 1M DTT per IP prep and place on ice.
• Prepare 100 µl 1X ChIP Buffer (10 µl 10X ChIP Buffer #7008 + 90 µl water) + 0.5 µl 200X PIC #7012 per IP prep and place on ice.

1. Extract nuclei by resuspending cells in 1 ml ice-cold 1X Buffer A + DTT + PIC per IP prep. Incubate on ice for 10 min. Mix by inverting the tube every 3 min.
2. Pellet nuclei by centrifugation at 2,000 x g in a benchtop centrifuge for 5 min at 4°C.
3. Remove supernatant and resuspend pellet in 1 ml ice-cold 1X Buffer B + DTT per IP prep.
4. Repeat centrifugation, remove supernatant, and resuspend pellet in 100 µl 1X Buffer B + DTT per IP prep.
5. Transfer sample to a 1.5 ml microcentrifuge tube, only fill up to 1 ml total per tube, add additional tubes if necessary.
6. Add 0.5 µl of Micrococcal Nuclease #10011 per IP prep, mix by inverting tube several times and incubate for 20 min at 37°C with shaking at up to 1,200 rpm to digest DNA to length of approximately 150-900 bp. If a thermomixer is not available, mix by inversion every 3 to 5 min.

   NOTE: 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). For example, HeLa nuclei digested with 0.5 µl Micrococcal Nuclease per 4 x 10⁶ cells gave the appropriate length DNA fragments (see Figure 4).

7. Stop digestion by adding 10 µl of 0.5 M EDTA #7011 per IP prep. Mix by gentle inversion and place the tube on ice for 1 to 2 min.
8. Pellet nuclei by centrifugation at 16,000 x g in a microcentrifuge for 1 min at 4°C and remove supernatant.
9. Resuspend the nuclear pellet in 100 µl of 1X ChIP Buffer + PIC per IP prep, only fill up to 500 µl total per tube, add additional tubes if necessary and incubate on ice for 10 min.
10. Sonicate up to 500 µl of lysate per 1.5 ml microcentrifuge tube with several pulses to break open the nuclear membrane. Incubate samples for 30 sec on wet ice between pulses. Optimal conditions required for complete lysis of nuclei can vary depending on different cell lines or different sonicators. For example, HeLa nuclei were completely lysed after 3 sets of 20-sec 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.
11. Clarify lysates by centrifugation at 9,400 x g in a microcentrifuge for 10 min at 4°C.

    NOTE: Only a minimal or barely visible pellet is expected following this step. If not, this may indicate insufficient sonication or homogenization, and additional sonication or homogenization may be required.

12. Transfer the supernatant to a new tube and aliquot 50 µl for Analysis of Chromatin Digestion and Concentration (Section III). (SAFE STOP) This 50 µl sample may be stored at -20°C overnight. The remaining fragmented chromatin should be stored at -80°C until further use.

III. Analysis of Chromatin Digestion and Concentration

1. To the 50 µl chromatin sample (from Step 12 in Section II), add 100 µl Nuclease-free Water #12931, 6 µl 5 M NaCl #7010, and 2 µl RNAse A (10 mg/ml) #7013. Gently vortex to mix and incubate the sample at 37°C for 30 min.
2. To the RNAse A-digested sample, add 2 µl Proteinase K (20 mg/ml) #10012. Gently vortex to mix and incubate the sample at 65°C for 2 h.
3. Purify DNA from the sample using a DNA purification spin column as described in Section VI. (SAFE STOP) DNA may be stored at -20°C for up to 6 months.
4. After purification of DNA, collect 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; see Figure 4).
5. To determine DNA concentration, measure a 2 µl aliquot by NanoDrop, Qubit or Picogreen assay. 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. Adding too little chromatin to the IP or over-digestion of chromatin may diminish signals in the PCR and sequencing-based analyses. Under-digestion of chromatin may lead to increased background signal and lower resolution. A protocol for optimization of chromatin digestion can be found in Appendix A.

IV. Chromatin Immunoprecipitation

For optimal ChIP results, use 10 µg of digested, cross-linked chromatin (as determined in Section III) per immunoprecipitation. This should be roughly equivalent to a single 100 µl IP prep from 4 x 10⁶ tissue culture cells. For high-abundance protein targets, the amount of chromatin per reaction can be reduced to 5 µg (equivalent to chromatin from 2 × 10⁶ cells); however, performance with reduced input may vary depending on the target.

Before starting:

(!) All buffer volumes should be increased proportionally based on the number of immunoprecipitations in the experiment.

• Warm Protease Inhibitor Cocktail (200X) #7012 (PIC). Make sure PIC is completely thawed.
• Warm 10X ChIP Buffer #7008 and ensure SDS is completely in solution.
• Thaw digested chromatin preparation (from Step 12 in Section II) and place on ice.
• Prepare low salt wash: 3 ml 1X ChIP Buffer (300 µl 10X ChIP Buffer #7008 + 2.7 ml water) per immunoprecipitation.
• Prepare high salt wash: 1 ml 1X ChIP Buffer (100 µl 10X ChIP Buffer #7008 + 900 µl water) + 70 µl 5M NaCl #7010 per immunoprecipitation.

1. For each immunoprecipitation, prepare a 500 µl diluted chromatin mix containing 10 µg of digested, cross-linked chromatin (from Step 12 in Section II), 2 µl of 200X PIC #7012, and sufficient 1X ChIP Buffer to bring the total volume to 500 µl (prepare 1X ChIP Buffer by diluting 10X ChIP Buffer #7008 tenfold with water, for example, 100 µl 10X ChIP Buffer + 900 µl water). Mix by gentle inversion and keep on ice.

   NOTE: When determining the number of immunoprecipitations, remember to include the positive control Histone H3 (D2B12) Rabbit Monoclonal Antibody (ChIP Formulated) #4620 and negative control Normal Rabbit IgG Antibody #2729 samples.

2. Collect a 10 µl sample of the diluted chromatin mix and transfer to a 1.5 ml microcentrifuge tube. This is your 2% Input Sample, which can be stored at -20°C until further use (Step 1 in Section V).
3. For each immunoprecipitation, transfer 500 µl of the diluted chromatin mix to a 1.5 ml microcentrifuge tube and add the immunoprecipitating antibody. The amount of antibody required per IP varies and should be determined by the user. For the positive control Histone H3 (D2B12) Rabbit Monoclonal Antibody (ChIP Formulated) #4620, add 10 µl to the IP sample. If using test antibodies from Cell Signaling Technology, please see recommended dilution listed on the datasheet or product webpage. For the negative control Normal Rabbit IgG #2729, add 2 µl (2 µg) to the IP sample or the same amount as the specific test antibody to the IP sample for a fair comparison.

   NOTE: Most antibodies from Cell Signaling Technology work optimally between 1 and 2 ug per IP sample. In the case where there are multiple samples with varying concentrations, it is best to match the negative control Normal Rabbit IgG #2729 to the highest antibody concentration.

4. Incubate IP samples for 4 h to overnight at 4°C with rotation.
5. Resuspend ChIP-Grade Protein G Magnetic Beads #9006 by gently vortexing. Immediately add 30 µl of ChIP-Grade Protein G Magnetic Beads #9006 to each IP reaction and incubate for 2 h at 4°C with rotation.
6. Pellet protein G magnetic beads in each immunoprecipitation by placing the tubes on a magnetic rack. Wait 1 to 2 min for solution to clear and then carefully remove supernatant.
7. Wash protein G magnetic beads by adding 1 ml of low salt wash to the beads and incubate at 4°C for 5 min with rotation. Repeat steps 6 and 7 two additional times for a total of 3 low salt washes.
8. Pellet protein G magnetic beads in each immunoprecipitation by placing the tubes on a magnetic rack. Wait 1 to 2 min for solution to clear and then carefully remove supernatant.
9. Add 1 ml of high salt wash to the beads and incubate at 4°C for 5 min with rotation.
10. Pellet protein G magnetic beads in each immunoprecipitation by placing the tubes on a magnetic rack. Wait 1 to 2 min for solution to clear and then carefully remove supernatant. Immediately proceed to Section V.

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

Before starting:

(!) All buffer volumes should be increased proportionally based on the number of immunoprecipitations in the experiment.

• Warm 2X ChIP Elution Buffer #7009 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 #7009 + 75 µl water) for each immunoprecipitation and the 2% input sample.

1. Add 150 µl of the 1X ChIP Elution Buffer to the 2% input sample tube and set aside at room temperature until Step 6.
2. Add 150 µl 1X ChIP Elution Buffer to each IP sample.
3. Elute chromatin from the antibody/protein G magnetic beads for 30 min at 65°C with gentle vortexing (1,200 rpm). A thermomixer works best for this step. Alternatively, mix the elutions by inversion every 3 to 5 min during the 65°C incubation period.
4. Pellet protein G magnetic beads by placing the tubes on a magnetic rack and wait 1 to 2 min for solution to clear.
5. Carefully transfer eluted chromatin supernatant to new tubes.
6. To all tubes, including the 2% input sample from Step 1, reverse cross-links by adding 6 µl 5M NaCl #7010 and 2 µl Proteinase K (20 mg/ml) #10012. Gently vortex to mix and incubate for 2 h at 65°C. This incubation can be extended overnight.
7. Immediately proceed to Section VI. (SAFE STOP) Alternatively, samples can be stored at -20°C for up to 4 days. However, to avoid formation of a precipitate, be sure to warm samples back to room temperature before adding DNA Binding Buffer (+ isopropanol) (Section VI, Step 1).

VI. DNA Purification Using Spin Columns:

Before starting:

• (!!) Add 11.25 ml of isopropanol to the DNA Binding Buffer #44364 before use. Skipping this step will result in complete loss of your DNA sample. This addition is required only once, prior to the first set of DNA purifications.
• (!!) Add 24 ml of ethanol (96-100%) to the DNA Wash Buffer #10008 before use. This step only has to be performed once prior to the first set of DNA purifications.
• Remove one DNA Purification Column and Collection Tube #10010 for each DNA sample from Section V.

1. Add 750 µl of DNA Binding Buffer (+ isopropanol) to each DNA sample and vortex briefly.
   • 5 volumes of DNA Binding Buffer (+ isopropanol) should be used for every 1 volume of sample.
2. Transfer 450 µl of each sample from Step 1 to a DNA purification spin 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. Place the spin column back into the collection tube.
5. Transfer the remaining 450 µl of each sample from Step 1 to the spin column in a collection tube. Repeat Steps 3 and 4.
6. Add 750 µl of DNA Wash Buffer (+ ethanol) to each spin column in a collection tube.
7. Centrifuge at 18,500 x g in a microcentrifuge for 30 sec.
8. Remove the spin column from the collection tube and discard the liquid. Place the spin column back into the collection tube.
9. Centrifuge at 18,500 x g in a microcentrifuge for 30 sec.
10. Discard collection tube and liquid. Retain the spin column.
11. Place each spin column into a clean 1.5 ml microcentrifuge tube and add 50 µl of DNA Elution Buffer #10009 to each.
12. Centrifuge at 18,500 x g in a microcentrifuge for 30 sec to elute and collect DNA.
13. Remove and discard DNA purification spin column. Eluate is now purified DNA. (SAFE STOP) Samples can be stored at -20°C for up to 6 months.

VII. Quantification of DNA by PCR:

Recommendations:

• Use filtered pipette tips to minimize risk of contamination.
• The control primers included in the kit are specific for the human or mouse RPL30 gene (#7014 and #7015 respectively) and can be used for either standard PCR or quantitative real-time PCR. If the user is performing ChIP 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 nonspecific 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

4. Start the following PCR reaction program:

Initial Denaturation:	95°C	5 min
Denature:	95°C	30 sec
Anneal:	62°C	30 sec
Extension:	72°C	30 sec
Final Extension:	72°C	5 min

5. Use 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 is 161 bp for human RPL30 #7014 and 159 bp for mouse RPL30 #7015.

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. (SAFE STOP) If necessary, cover the plate with aluminum foil to avoid light and store at 4°C up to 4 hours or -20°C overnight until the machine is ready for use.

Reagent	Volume for 1 PCR Reaction (18 µl)
Nuclease-free H2O	6 µl
5 µM RPL30 Primers	2 µl
SimpleChIP® Universal qPCR Master Mix #88989	10 µl

4. 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.

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. Background signals are typically below 0.1% of input, and a signal-to-noise ratio greater than 3 is considered ideal.

Percent Input = 2% x 2^{(C[T] 2%Input Sample - C[T] IP Sample)}

C[T] = C_T = C_q = Threshold cycle of PCR reaction

VIII. NG-Sequencing Library Construction

The immuno-enriched DNA samples prepared with this kit are directly compatible with next-generation sequencing (NG-seq). For downstream NG-seq DNA library construction, use a DNA library preparation protocol or kit compatible with your downstream sequencing platform. For sequencing on Illumina Systems platforms, we recommend DNA Library Prep Kit for Illumina Systems (ChIP-seq, CUT&RUN) #56795 and its associated index primers Multiplex Oligos for Illumina Systems (Single Index Primers) (ChIP-seq, CUT&RUN) #29580 or Multiplex Oligos for Illumina Systems (Dual Index Primers) (ChIP-seq, CUT&RUN) #47538.

Recommendations:

• For transcription factor or co-factor ChIP-seq, use at least 5 ng of ChIP-enriched DNA and amplification of the adaptor-ligated DNA with 10 cycles of PCR.
• For total histone and histone modifications, or input samples, start with 50 ng of ChIP-enriched DNA and amplification of the adaptor-ligated DNA with 6 cycles of PCR.
• For library construction of ChIP-enriched DNA for all target types, perform cleanup of adaptor-ligated DNA without size selection.
• After DNA library construction, check the DNA library for presence of adaptor dimers (~140 bp) using a BioAnalyzer system, TapeStation system, Fragment Analyzer system, or by agarose gel electrophoresis with 50-100 ng DNA on a 2% agarose TAE gel. If adaptor dimers are present in the DNA library, repeat cleanup of PCR amplified material.
• The quality of the library can also be confirmed using qPCR and primer sets to known positive and negative target loci. Positive primer pairs should still give the same high signal compared to negative primers as seen in the original qPCR analysis of ChIP-enriched DNA.
• After final cleanup and quality checks, prepare final purified library samples at 2 to 10 nM for high throughput sequencing.

APPENDIX A: Optimization of Chromatin Digestion

Optimal conditions for the digestion of cross-linked chromatin DNA to 150-900 base pairs in length is highly dependent on the ratio of Micrococcal Nuclease to the amount of cells used in the digest. Below is a protocol for determination of the optimal digestion conditions for a specific cell type.

1. Prepare cross-linked nuclei from 2 X 10⁷ cells (equivalent of 5 IP preps), as described in Sections I, II, and III. Stop after Step 5 of Section II and proceed as described below.
2. Transfer 100 µl of the nuclei preparation into 5 individual 1.5 ml microcentrifuge tubes and place on ice.
3. Add 3 µl Micrococcal Nuclease #10011 to 27 µl of 1X Buffer B + DTT (1:10 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 each digestion by adding 10 µl of 0.5 M EDTA #7011. Mix by gentle inversion and place the tube on ice for 1 to 2 min.
6. Pellet nuclei by centrifugation at 16,000 x g in a microcentrifuge for 1 min at 4°C and remove supernatant.
7. Resuspend nuclear pellet in 200 µl of 1X ChIP Buffer + PIC. Incubate on ice for 10 min.
8. Sonicate lysates with several pulses to break open the nuclear membrane. Incubate samples for 30 sec on wet ice between pulses. Optimal conditions required for complete lysis of nuclei can vary depending on different cell lines or different sonicators. For example, HeLa nuclei were completely lysed after 3 sets of 20-sec 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 9,400 x g in a microcentrifuge for 10 min at 4°C.

   NOTE: Only a minimal or barely visible pellet is expected following this step. If not, this may indicate insufficient sonication or homogenization, and additional sonication or homogenization may be required.

10. Transfer 50 µl of each of the sonicated lysates to new 1.5 ml microcentrifuge tubes.
11. To each 50 µl sample, add 100 µl Nuclease-free Water #12931, 6 µl 5 M NaCl #7010 and 2 µl RNAse A (10 mg/ml) #7013. Gently vortex to mix and incubate samples at 37°C for 30 min.
12. To each RNAse A-digested sample, add 2 µl Proteinase K (20 mg/ml) #10012. Gently vortex to mix and incubate samples at 65°C for 2 h.
13. Purify DNA from samples using DNA purification spin columns as described in Section VI.
14. Use 10 µl of each sample and determine DNA fragment size by electrophoresis on a 1% agarose gel with a 100 bp DNA marker.
15. Observe which of the digestion conditions produces DNA in the desired range of 150-900 base pairs (1 to 5 nucleosomes, see Figure 4). The volume of diluted Micrococcal Nuclease that produces the desired size of DNA fragments using this optimization protocol is equivalent to 10 times the volume of Micrococcal Nuclease #10011 that should be added to one immunoprecipitation preparation (4 X 10⁶ tissue culture 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 0.5 µl of Micrococcal Nuclease #10011 should be added to one IP prep during the digestion of chromatin in Section II.
16. 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. Alternatively, the digestion time can be changed to increase or decrease the extent of DNA fragmentation.

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 at least 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. Too many cells or not enough Micrococcal Nuclease was added to the chromatin digestion.	Perform a time course at a fixed formaldehyde concentration. Shorten the time of cross-linking to 10 min or less. 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. PCR amplified regions may span nucleosome-free regions. Not enough chromatin added to the IP or chromatin is over-digested.	Add more DNA to the PCR reaction or increase the number of amplification cycles. Optimize the PCR conditions for experimental primer sets using purified DNA from cross-linked and digested chromatin. Design a different primer set and decrease the length of amplicon to less than 150 bp (see primer design recommendations in Section VII). For optimal ChIP results add 5-10 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. Incomplete elution of chromatin from Protein G beads.	Be sure to add 5-10 µg of chromatin and 10 µl of histone H3 antibody to each IP reaction and incubate with antibody over-night and an additional 2 h after adding 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 or not enough chromatin added to the IP reaction. Alternatively, too much antibody added to the IP reaction. Too much DNA added to the PCR reaction or too many cycles of amplification.	Add no more than 15 µg of chromatin and 10 µl of histone H3 antibody to each IP reaction. Shorten the amount of normal rabbit IgG to 1 µl per IP. 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. Not enough antibody added to the IP reaction. Antibody does not work for IP.	Add more DNA to the PCR reaction or increase the number of amplification cycles. Typically a range of 1 to 5 µg of antibody is added to the IP reaction; however, the exact amount depends greatly on the individual antibody. Increase the amount of antibody added to the IP. Find an alternate antibody source.

APPENDIX C: Online Resources

For the visual protocol, seminars, blogs, FAQs, and further information on the ChIP and ChIP-seq assays, please visit our ChIP & ChIP-seq Resource Center at https://www.cellsignal.com/applications/chip-and-chip-seq