SimpleChIP® Plus Sonication Chromatin Immunoprecipitation Protocol (Magnetic Beads)

Specific for product: SimpleChIP^® Plus Sonication Chromatin IP Kit #56383.

I. Tissue Cross-linking and Sample Preparation

When harvesting tissue, remove unwanted material from the sample, such as fat and necrotic material. Tissue can be processed and cross-linked immediately or frozen on dry ice for processing later. For optimal ChIP results, use 25 mg of tissue for each immunoprecipitation to be performed. An additional 5 mg of tissue should be processed for Analysis of Chromatin Digestion and Concentration (Section IV). The chromatin yield does vary between tissue types, and some tissues may require more than 25 mg for each immunoprecipitation.

One chromatin preparation is defined as 100 to 150 mg of tissue. This recommended amount of tissue accounts for potential low yield with some tissue types and also ensures efficient chromatin fragmentation during sonication. Please see Appendix A for more information regarding the expected chromatin yield for different types of tissue.

Before starting:

• Warm Protease Inhibitor Cocktail (200X) #7012 (PIC) and Glycine Solution (10X) #7005. Make sure PIC is completely thawed.
• Prepare 3 ml of Phosphate Buffered Saline (PBS) #9872 + 15 µl 200X PIC #7012 per chromatin preparation and place on ice.
• Prepare 1 ml of 1X ChIP Sonication Cell Lysis Buffer (0.5 ml ChIP Sonication Cell Lysis Buffer (2X) #96529 + 0.5 ml water) + 5 µl 200X PIC #7012 per chromatin preparation and place on ice.
• Prepare 27 µl of 37% formaldehyde or 62.5 µl of 16% Formaldehyde, Methanol-Free #12606 per chromatin preparation and keep at room temperature. Use fresh formaldehyde that is not past the manufacturer's expiration date.

1. Weigh the fresh or frozen tissue sample. Collect 100 to 150 mg of tissue per chromatin preparation (at least 75 mg of tissue is required for one experiment in order to include positive and negative controls).
2. Place tissue sample in a petri dish and finely mince into 1-2 mm cubes using a clean scalpel or razor blade. Keep the dish on ice. It is important to keep the tissue cold to avoid protein degradation.
3. Transfer minced tissue to a 15 ml conical tube and add 1 ml of ice-cold PBS + PIC per chromatin preparation.
4. To crosslink proteins to DNA, add formaldehyde to a final concentration of 1% by adding 27 µl of 37% formaldehyde or 62.5 µl of 16% Formaldehyde, Methanol-Free #12606 per 1 ml of PBS + PIC. Invert to mix and incubate at room temperature for at least 10 min.

   NOTE: For histone modification ChIP, 10 min fixation is sufficient; however, for transcription factor ChIP, we recommend fixation for 10 to 30 min, and for transcription cofactor ChIP, we recommend fixation for 30 min (see Figure 7 in Appendix B).

5. Stop cross-linking by adding 100 µl of Glycine Solution (10X) #7005 per 1 ml of PBS + PIC. Invert to mix and incubate for 5 min at room temperature.
6. Centrifuge tissue at 1,200 x g for 5 min at 4°C.
7. Remove supernatant and wash with 1 ml of ice-cold PBS + PIC per chromatin preparation.
8. Centrifuge at 1,200 x g for 5 min at 4°C.
9. Repeat steps 7 and 8 one additional time.
10. Remove supernatant and proceed to step 11. (SAFE STOP) Alternatively, samples may be stored at -80°C before disaggregation for up to 3 months.
11. Resuspend tissue in 1 ml 1X ChIP Sonication Cell Lysis Buffer + PIC per chromatin preparation.
12. Transfer tissue suspension to a Dounce homogenizer using a cut pipet tip.
13. Using a tight fitting pestle (Type A), disaggregate tissue pieces with 20 strokes or until no chunks of tissue are observed.
14. Transfer cell suspension to a 1.5 ml tube and immediately proceed to Nuclei Preparation and Chromatin Fragmentation (Section III).

II. Cell Culture Cross-linking and Sample Preparation

For optimal ChIP results, use approximately 4 x 10⁶ cells for each immunoprecipitation to be performed. For HCT 116 cells, this is equivalent to 1/3 of a 15 cm culture dish containing cells that are 90% confluent in 20 ml of growth medium. An additional 1 x 10⁶ cells should be processed for Analysis of Chromatin Digestion and Concentration (Section IV). 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.

One chromatin preparation is defined as 1.5 x 10⁷ to 2 x 10⁷ cells. This recommended cell number accounts for potential low yield with some cell types and also ensures efficient chromatin fragmentation during sonication.

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.
• Prepare 1 ml of 1X ChIP Sonication Cell Lysis Buffer (0.5 ml ChIP Sonication Cell Lysis Buffer (2X) #96529 + 0.5 ml water) + 5 µl 200X PIC #7012 per chromatin preparation.

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:
   1. Transfer cells to a 50 ml conical tube, centrifuge at 2,000 x g for 5 min at 4°C, and wash the pellet two times with 20 ml ice-cold PBS. Remove PBS and proceed to step 3b. (SAFE STOP) Alternatively, cell pellets can be frozen on dry ice and stored at -80°C for up to 3 months.
   2. Resuspend up to 2 x 10⁷ cells per 1 ml of 1X ChIP Sonication Cell Lysis Buffer + PIC per chromatin preparation. Only fill up to 1 ml total per tube, add additional tubes if necessary. Immediately proceed to Nuclei Preparation and Chromatin Fragmentation (Section III).
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 for 5 min at 4°C. Remove PBS and proceed to step 4d. (SAFE STOP) Alternatively, cell pellets can be frozen on dry ice and stored at -80°C for up to 3 months.
   4. Resuspend up to 2 x 10⁷ cells in 1 ml of 1X ChIP Sonication Cell Lysis Buffer + PIC per chromatin preparation. Only fill up to 1 ml total per tube, add additional tubes if necessary. Immediately proceed to Nuclei Preparation and Chromatin Fragmentation (Section III).

III. Nuclei Preparation and Chromatin Digestion

One chromatin preparation is defined as 100 to 150 mg of tissue or 1.5 x 10⁷ to 2 x 10⁷ tissue culture cells. Multiple chromatin preparations can be performed simultaneously, as long as the amounts of buffers are scaled appropriately and sonication is performed on 1 ml samples. The number of cells and volume of sample used for sonication is critical for generation of appropriately sized chromatin fragments.

Before starting:

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

• Warm Protease Inhibitor Cocktail (200X) #7012 (PIC). Make sure it is completely thawed prior to use.
• Prepare 1 ml of 1X ChIP Sonication Cell Lysis Buffer (0.5 ml ChIP Sonication Cell Lysis Buffer (2X) #96529 + 0.5 ml water) + 5 µl 200X PIC #7012 per chromatin preparation.
• Prepare 1 ml ChIP Sonication Nuclear Lysis Buffer #28778 + 5 µl 200X PIC #7012 per chromatin preparation.

1. Incubate cell suspension in 1X ChIP Sonication Cell Lysis Buffer + PIC from Section I or Section II on ice for 10 min.
2. Pellet cells at 5,000 x g for 5 min at 4°C. Remove supernatant and resuspend the pellet a second time in 1 ml ice-cold 1X ChIP Sonication Cell Lysis Buffer + PIC per chromatin preparation.
3. Incubate cell suspension on ice for 5 min. Pellet cells at 5,000 x g for 5 min at 4°C.
4. Resuspend nuclei in 1 ml ice-cold ChIP Sonication Nuclear Lysis Buffer + PIC per chromatin preparation. Aliquot 1 ml of nuclei suspension per chromatin preparation to an appropriately sized tube for sonication and incubate on ice for 10 min.

   NOTE: Do not exceed 1 ml of nuclei suspension per sonication tube when processing multiple chromatin preparations.

   NOTE: Cross-linked cells and nuclei may not be completely lysed until sonication.

5. Fragment chromatin by sonication. Sonication conditions may need to be determined empirically by testing different sonicator power settings and durations. Optimal sonication conditions will generate chromatin in which 60 to 90% of the chromatin fragments are smaller than 1kb. Longer cross-linking times may decrease the proportion of fragments being smaller than 1 kb to 30 to 60% (see Figure 7 in Appendix B and Figure 8 in Appendix C). Use the minimal number of sonication cycles required to generate the desired length of chromatin fragments, as over-sonication can result in reduction or loss of signal due to harsh treatment of the chromatin.
   • For each sonication sample, we recommend using 100 to 150 mg of tissue or 1.5 x 10⁷ to 2 x 10⁷cells per 1 ml ChIP Sonication Nuclear Lysis Buffer. Sonicating in larger volumes and/or using greater concentration of cells will decrease the efficiency of chromatin fragmentation.
   • Be sure to keep the chromatin sample cool during sonication by keeping the tube containing the chromatin in an ice water bath during and in between the sonication steps. Do not allow the probe to touch the bottom or wall of the tube. Stop sonication and adjust the position of the tube if the chromatin sample foams during sonication.
   • Using a Branson Digital Sonifier D250 probe sonicator with a 1/8-inch Micro Tip, 8 min of 1 sec on/1 sec off sonication cycle (4 min of sonication time) at 50% amplitude typically gives good fragmentation and chromatin IP efficiency.
6. Clarify lysates by centrifugation at 21,000 x g in a microcentrifuge for 10 min at 4°C.
7. Transfer the supernatant to a new tube and aliquot 50 µl for Analysis of Chromatin Digestion and Concentration (Section IV). (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.

IV. Analysis of Chromatin Digestion and Concentration

1. To the 50 µl chromatin sample (from Step 7 in Section III), 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 hr.
3. Purify DNA from the sample using a DNA purification spin column as described in Section VII. (SAFE STOP) DNA may be stored at -20°C for up to 6 months.
4. After purification of DNA, collect a 20 µl sample and determine DNA fragment size by electrophoresis on a 1% agarose gel. A DNA smear ranging from 200 bp to several kb is expected (see Figure 7 in Appendix C). Approximately 60 to 90% of total DNA fragments should be less than 1 kb. Refer to Appendix B for optimization of sonication conditions.
5. To determine DNA concentration, measure a 2 µl aliquot by NanoDrop, Qubit or Picogreen assay. Ideally, DNA concentration should be between 50 and 100 µg/ml.

V. Chromatin Immunoprecipitation

For optimal ChIP results, use 10 µg of sonicated, cross-linked chromatin (as determined in Section IV) per immunoprecipitation. This should be roughly equivalent to the chromatin amount derived from 25 mg of disaggregated tissue or 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 x 10⁶ cells or 12.5 mg tissue); 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 fragmented chromatin preparation (from Step 7 in Section III) 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 Section III), 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 the mixture on ice. For efficient immunoprecipitation, chromatin must be diluted in 1X ChIP Buffer at a minimum dilution ratio of 1:4. If more than 100 µl of chromatin sample (from Section III) is required to obtain 10 µg of chromatin, it is recommended to use an IP volume greater than 500 µl to maintain at least a 1:4 dilution. In this case, the increased IP volume (still containing 10 µg of chromatin DNA) does not require additional antibody in Step 3.

   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 2% aliquot of the diluted chromatin (for example, 10 µl from 500 µl total volume) and transfer it to a microcentrifuge tube. This is your 2% Input Sample, which can be stored at -20°C until further use (Step 1 in Section VI).
3. For each immunoprecipitation, transfer the appropriate volume of diluted chromatin mix containing 10 µg of chromatin DNA to a 1.5 ml microcentrifuge tube (for example, 500 µl) and add the immunoprecipitating antibody. The amount of antibody required per IP may vary and should be determined by the user.

   NOTE: For the positive control Histone H3 (D2B12) Rabbit Monoclonal Antibody (ChIP Formulated) #4620, add 10 µl to the IP sample containing 10 µg of chromatin DNA, regardless of IP volume.

   NOTE: If using test antibodies from Cell Signaling Technology, refer to the recommended dilution on the datasheet or product webpage. Although the recommended antibody ratio is based on 500 µl of diluted chromatin containing 10 µg of chromatin DNA, increasing the IP reaction volume (while keeping chromatin at 10 µg) does not require additional antibody. The ratio of antibody volume to chromatin amount is more critical than the antibody volume relative to the total IP volume.

   NOTE: For the negative control Normal Rabbit IgG #2729, add 2 µl (2 µg) to the IP sample containing 10 µg of chromatin DNA, or use the same amount as the test antibody for a fair comparison. Most antibodies from Cell Signaling Technology work optimally between 1 and 2 µg 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 separation 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 5 and 6 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 separation 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 separation rack. Wait 1 to 2 min for solution to clear and then carefully remove supernatant. Immediately proceed to Section VI.

VI. 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 7.
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; however a 65°C water bath with frequent mixing is also sufficient.
4. Briefly centrifuge samples at 10,000 x g for 10 sec to collect evaporation from the microcentrifuge tube cap.
5. Pellet protein G magnetic beads by placing the tubes on a magnetic separation rack and wait 1 to 2 min for solution to clear.
6. Carefully transfer eluted chromatin supernatant to new tubes.
7. 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.
8. Immediately proceed to Section VII. (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 VII, Step 1).

VII. 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.
• Prepare one DNA Purification Column and Collection Tube #10010 for each DNA sample from Section VI.

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 DNA 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 DNA 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 the 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 spin column. Eluate is now purified DNA. (SAFE STOP) Samples can be stored at -20°C for up to 6 months.

VIII. 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 PCR tubes or PCR plates compatible with the model of PCR machine to be used. 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:

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

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. Setup 2-3 replicates for each PCR reaction. Add enough reagents 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

IX. 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, 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: Expected Chromatin Yield

When harvesting cross-linked chromatin from tissue samples, the yield of chromatin can vary significantly between tissue types. The table in this section provides a range for the expected yield of chromatin from 100 mg of tissue compared to 2 x 10⁷ HCT 116 cells, and the expected DNA concentration, as determined in Section IV of the protocol. For optimal ChIP results, we recommend using 10 µg of digested, cross-linked chromatin per immunoprecipitation; therefore, some tissues may require harvesting more than 25 mg per immunoprecipitation.

Tissue/Cell	Total Chromatin Yield	Expected DNA Concentration
Liver	10-15 µg per 25 mg tissue	100-150 µg/ml
Brain	2-5 µg per 25 mg tissue	20-50 µg/ml
Heart	2-5 µg per 25 mg tissue	20-50 µg/ml
HCT 116	75-150 µg per 2 x 107 cells	75-150 µg/ml

Appendix B: Optimization of Formaldehyde Fixation

Transcription factors and cofactors bind to chromatin DNA more loosely than histone proteins. As a result, they tend to dissociate from chromatin during sonication. Additional fixation time can result in increased capture of transcription factors and cofactors in the ChIP assay, especially with tissue samples. As shown in Figure 7, increasing the fixation time from 10 min to 30 min may reduce chromatin fragmentation size (left panel), but it significantly enhances enrichment of both cofactors RING1B and SUZ12 in heart tissue, as indicated by ChIP-qPCR (middle and right panels).

Typically, 10 min fixation is sufficient for histone modification ChIP with both cell and tissue samples, whereas transcription factors and cofactors may require additional fixation up to 30 min, especially with tissue samples.

FIGURE 7. Mouse heart (H), brain (B), and liver (L) were cross-linked for 10 min or 30 min, as indicated (left panel). The chromatin was prepared and sonicated, DNA was purified and separated by electrophoresis on a 1% agarose gel. In the ChIP-qPCR assay (middle and right panels), chromatin immunoprecipitations were performed with either RING1B (D22F2) Rabbit Monoclonal Antibody #5694 or SUZ12 (D39F6) Rabbit Monoclonal Antibody #3737. The enriched DNA was quantified by real-time PCR using SimpleChIP^® Mouse HoxD10 Exon 1 Primers #7429, SimpleChIP^® Mouse HoxA1 Promoter Primers #7341, and SimpleChIP^® Mouse GAPDH Intron 2 Primers #8986. The amount of immunoprecipitated DNA in each sample is represented as normalized signal to the negative GAPDH locus (equivalent to one).

Appendix C: Optimization of Chromatin Fragmentation

Optimal conditions for the fragmentation of cross-linked chromatin DNA is highly dependent on the number of cells, volume of sample, length of sonication, and sonicator power setting used. For each sonication sample, we recommend using 100-150 mg of tissue or 1.5 x 10⁷ to 2 x 10⁷ cells per 1 ml ChIP Sonication Nuclear Lysis Buffer. Below is a protocol for determination of the optimal sonication conditions for a specific tissue or cell type.

1. Prepare cross-linked nuclei from 100 to 150 mg of tissue or 1.5 x 10⁷ to 2 x 10⁷ cells, as described in Sections I, II, and III. Stop after Step 4 of Section III and proceed as described below.
2. Fragment chromatin by sonication. Optimal sonication conditions can be determined for a given sonicator by varying the number of rounds or duration of sonication at a given power setting (see Step 5 in Section III for optimal power setting using a Branson Digital Sonifier 250 probe sonicator). To determine optimal sonication conditions, set up a sonication time-course experiment and collect 50 µl samples of chromatin after a given round or duration of sonication. For example, take chromatin samples after each 1 to 2 min of sonication.
3. Clarify chromatin samples by centrifugation at 21,000 x g in a microcentrifuge for 10 min at 4°C.
4. Transfer supernatants to new 1.5 ml microcentrifuge tubes and 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.
5. 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 hr.
6. Purify DNA from samples using DNA purification spin columns as described in Section VII.
7. Use 20 µl of each sample and determine DNA fragment size.
8. Choose the sonication conditions that generate optimal DNA fragment size and use for chromatin preparation in Step 5 of Section III. If optimal sonication conditions are not achieved, increase or decrease the power setting of the sonicator and repeat the sonication time course.

   NOTE: Optimal sonication conditions can vary with different sample types and fixation times. Use the minimal number of sonication cycles required to generate the desired length of chromatin fragments. Over sonication, as indicated by more than 80% of total DNA fragments less than 500 bp, can result in excessive damage to the chromatin and result in lower immunoprecipitation efficiency (see Figure 8, right panel).

• For sonication of cells fixed for 10 min, optimal sonication conditions will generate a DNA smear with approximately 90% of total DNA fragments less than 1 kb (see Figure 8, left panel). Increasing the fixation time to 30 min will limit fragmentation, generating a DNA smear with approximately 60% of total DNA fragments less than 1 kb.
• For sonication of tissues fixed for 10 min, optimal sonication conditions will generate a DNA smear with approximately 60% of total DNA fragments less than 1 kb. Increasing the fixation time to 30 min will limit fragmentation, generating a DNA smear with approximately 30% of total DNA fragments less than 1 kb (see Figure 7, left panel).

FIGURE 8. HCT 116 cells were cross-linked for 10 min and sonicated for the time indicated (left panel). DNA was purified as described in Section IV of the SimpleChIP^® Plus Sonication Chromatin IP Kit #56383, and 20 µl of purified DNA was separated by electrophoresis on a 1% agarose gel. As shown in the left panel, increasing cycles of sonication reduces the size of chromatin fragments. Chromatin immunoprecipitations were performed with either Non-phospho (Active) β-Catenin (Ser33/37/Thr41) (D13A1) Rabbit Monoclonal Antibody #8814, TCF4/TCF7L2 (C48H11) Rabbit Monoclonal Antibody #2569, or Normal Rabbit IgG #2729 using SimpleChIP^® Plus Sonication Chromatin IP Kit #56383. The enriched DNA was quantified by real-time PCR using SimpleChIP^® Human CaMK2D Intron 3 Primers #5111 and SimpleChIP^® Human α Satellite Repeat Primers #4486. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin (equivalent to 1; right panel). As shown, 4 min of chromatin sonication is optimal when using a Branson Digital Sonifier D250 probe sonicator with a 1/8 inch Micro Tip. Over-sonication significantly impairs enrichment of both cofactor beta-catenin and transcription factor TCF4/TCF7L2 containing chromatin.

APPENDIX D: Troubleshooting Guide

Problem	Possible Causes	Recommendation
1. Concentration of the fragmented chromatin is too low.	Cell/nuclear lysis is incomplete. Not enough cells/tissues were used for the chromatin preparation.	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.
2. Chromatin is under-fragmented and fragments are too large (>50% above 1.5kb).	Cells/tissues may have been over cross-linked. Too many cells/tissues were processed.	Shorten the crosslinking time to be within the 10-30 minute range. Reduce the amount of cell/tissues per sonication. Conduct sonication time course.
3. Chromatin is over-fragmented (>90% under 300 bp).	Sonication condition is too harsh.	Conduct a sonication time course to find a minimum output/duration to achieve appropriate sonication.
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-sonicated	Add more DNA to the PCR reaction or increase the number of amplification cycles. Optimize the PCR conditions for experimental primer set using purified DNA from cross-linked and sonicated chromatin. 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 hr 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. Reduce 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 cannot 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 E: 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