DNMT3A (D2H4B) Rabbit mAb #32578

Chromatin IP

Specific for product: SimpleChIP^® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005.

Required Reagents

Reagents Included:

1. Glycine Solution (10X) #7005
2. Buffer A (4X) #7006
3. Buffer B (4X) #7007
4. ChIP Buffer (10X) #7008
5. ChIP Elution Buffer (2X) #7009
6. 5 M NaCl #7010
7. 0.5 M EDTA #7011
8. ChIP-Grade Protein G Magnetic Beads #9006
9. DNA Binding Buffer #10007
10. DNA Wash Buffer (add 4x volume ethanol before use) #10008
11. DNA Elution Buffer #10009
12. DNA Purification Columns and Collection Tubes #10010
13. Protease Inhibitor Cocktail (200X) #7012
14. RNAse A (10 mg/ml) #7013
15. Micrococcal Nuclease #10011
16. Proteinase K (20 mg/ml) #10012
17. SimpleChIP^® Human RPL30 Exon 3 Primers 1 #7014
18. SimpleChIP^® Mouse RPL30 Intron 2 Primers 1 #7015
19. Histone H3 (D2B12) XP^® Rabbit mAb (ChIP Formulated) #4620
20. Normal Rabbit IgG #2729
21. DTT (Dithiothreitol) #7016

Reagents Not Included:

1. Magnetic Separation Rack #7017 / 14654
2. Phosphate Buffered Saline (PBS-1X) pH7.2 (Sterile) #9872
3. Nuclease-free Water #12931
4. Ethanol (96-100%)
5. Formaldehyde (37% Stock)
6. SimpleChIP^® Universal qPCR Master Mix #88989

I. Tissue Cross-linking and Sample Preparation

When harvesting tissue, remove unwanted material such as fat and necrotic material from the sample. Tissue can then be processed and cross-linked immediately, or frozen on dry ice and stored at -80°C for processing later. For optimal chromatin yield and ChIP results, use 25 mg of tissue for each immunoprecipitation to be performed. The chromatin yield does vary between tissue types and some tissues may require more than 25 mg for each immunoprecipitation. Please see Appendix A for more information regarding the expected chromatin yield for different types of tissue. One additional chromatin sample should be processed for Analysis of Chromatin Digestion and Concentration (Section IV). If desired, five additional chromatin samples should be processed for Optimization of Chromatin Digestion (Appendix B).

Before starting:

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

• Remove and warm 200X Protease Inhibitor Cocktail (PIC) and 10X Glycine Solution. Make sure PIC is completely thawed.
• Prepare 3 ml of Phosphate Buffered Saline (PBS) + 15 µl 200X PIC per 25 mg of tissue to be processed and place on ice.
• Prepare 45 µl of 37% formaldehyde per 25 mg of tissue to be processed and keep at room temperature. Use fresh formaldehyde that is not past the manufacturer's expiration date.

A. Cross-linking

1. Weigh the fresh or frozen tissue sample. Use 25 mg of tissue for each IP to be performed (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 60 mm or 100 mm dish and finely mince using a clean scalpel or razor blade. Keep 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.
4. Add 1 ml of PBS + PIC per 25 mg tissue to the conical tube.
5. To crosslink proteins to DNA, add 45 µl of 37% formaldehyde per 1 ml of PBS + PIC and rock at room temp for 20 min. Final formaldehyde concentration is 1.5%.
6. Stop cross-linking by adding 100 µl of 10X Glycine per 1 ml of PBS + PIC and mix for 5 min at room temperature.
7. Centrifuge tissue at 500 x g in a benchtop centrifuge for 5 min at 4°C.
8. Remove supernatant and wash one time with 1 ml PBS + PIC per 25 mg tissue.
9. Repeat centrifugation at 500 x g in a benchtop centrifuge for 5 min at 4°C.
10. Remove supernatant and resuspend tissue in 1 ml PBS + PIC per 25 mg tissue and store on ice. Disaggregate tissue into single-cell suspension using a Medimachine (Part B) or Dounce homogenizer (Part C). (SAFE STOP) Alternatively, samples may be stored at -80°C before disaggregation for up to 3 months.

B. Tissue Disaggregation Using Medimachine from BD Biosciences (part #340587)

1. Cut off the end of a 1000 µL pipette tip to enlarge the opening for transfer of tissue chunks.
2. Transfer 1 ml of tissue resuspended in PBS + PIC into the top chamber of a 50 mm medicone (part #340592).
3. Grind tissue for 2 min according to manufacturer's instructions.
4. Collect cell suspension from the bottom chamber of the medicone using a 1 ml syringe and 18 gauge blunt needle. Transfer cell suspension to a 15 ml conical tube and place on ice.
5. Repeat steps 2 to 4 until all the tissue is processed into a homogenous suspension.
6. If more grinding is necessary, add more PBS + PIC to tissue. Repeat steps 2 to 5 until all tissue is ground into a homogeneous suspension.
7. Check for single-cell suspension by microscope (optional).
8. Centrifuge cells at 2,000 x g in a bench top centrifuge for 5 min at 4°C.
9. Remove supernatant from cells and continue with Nuclei Preparation and Chromatin Digestion (Section III).

C. Tissue Disaggregation Using a Dounce Homogenizer

1. Transfer tissue resuspended in PBS + PIC to a Dounce homogenizer.
2. Disaggregate tissue pieces with 20-25 strokes. Check for single-cell suspension by microscope (optional).
3. Transfer cell suspension to a 15 ml conical tube and centrifuge at 2,000 x g in a benchtop centrifuge for 5 min at 4°C.
4. Remove supernatant from cells and continue with Nuclei Preparation and Chromatin Digestion (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 (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 IV). Since every cell type is different, we recommend including one extra dish of cells in 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 (or 20 ml suspension cells) used.

• Remove and warm 200X Protease Inhibitor Cocktail (PIC) #7012 and 10X Glycine Solution #7005. Make sure PIC is completely thawed.
• Prepare 2 ml of Phosphate Buffered Saline (PBS) + 10 µl 200X PIC per 15 cm dish (or 20 ml suspension cells) to be processed and place on ice.
• Prepare 40 ml of PBS per 15 cm dish (or 20 ml suspension cells) to be processed and place on ice.
• Prepare 540 µl of 37% formaldehyde per 15 cm dish (or 20 ml 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 540 µl of 37% formaldehyde to each 15 cm culture dish containing 20 ml medium. For suspension cells, add 540 µl of 37% formaldehyde to cells suspended in 20 ml medium (for optimal fixation of suspension cells, cell density should be less than 0.5 x 10⁶ cells/ml at fixation). Swirl briefly to mix and incubate 10 min at room temperature. Final formaldehyde concentration is 1%. Addition of formaldehyde may result in a color change of the medium.
2. Add 2 ml of 10X glycine to each 15 cm dish containing 20 ml medium, swirl briefly to mix, and incubate 5 min 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 500 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 III).
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.
5. Add 2 ml ice-cold PBS + PIC to each 15 cm dish. Scrape cells into cold buffer. Combine cells from all culture dishes into one 15 ml conical tube.
6. 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 III). (SAFE STOP) Alternatively samples may be stored at -80°C for up to 3 months.

III. Nuclei Preparation and Chromatin Digestion

Before starting

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

• Remove and warm 200X Protease Inhibitor Cocktail (PIC) #7012. 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.

  (!!) IMPORTANT: Once in solution, store 1M DTT at -20°C.

• Remove and 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 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 per IP prep and place on ice.

1. Resuspend cells in 1 ml ice-cold 1X Buffer A + DTT + PIC per IP prep. Incubate on ice for 10 min. Mix by inverting tube every 3 min.
2. Pellet nuclei by centrifugation at 2,000 x g in a benchtop centrifuge for 5 min at 4°C. Remove supernatant and resuspend pellet in 1 ml ice-cold 1X Buffer B + DTT per IP prep. Repeat centrifugation, remove supernatant, and resuspend pellet in 100 µl 1X Buffer B +DTT per IP prep. Transfer sample to a 1.5 ml microcentrifuge tube, up to 1 ml total per tube.
3. 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 frequent mixing to digest DNA to length of approximately 150-900 bp. Mix by inversion every 3 to 5 min. The amount of Micrococcal Nuclease required to digest DNA to the optimal length may need to be determined empirically for individual tissues and cell lines (see Appendix B). HeLa nuclei digested with 0.5 µl Micrococcal Nuclease per 4 x 10⁶ cells and mouse liver tissue digested with 0.5 µl Micrococcal Nuclease per 25 mg of tissue gave the appropriate length DNA fragments.
4. Stop digest by adding 10 µl of 0.5 M EDTA #7011 per IP prep and placing tube on ice for 1-2 min.
5. Pellet nuclei by centrifugation at 16,000 x g in a microcentrifuge for 1 min at 4°C and remove supernatant.
6. Resuspend nuclear pellet in 100 µl of 1X ChIP Buffer + PIC per IP prep and incubate on ice for 10 min.
7. Sonicate up to 500 µl of lysate per 1.5 ml microcentrifuge tube with several pulses to break nuclear membrane. Incubate samples for 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-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.
8. Clarify lysates by centrifugation at 9,400 x g in a microcentrifuge for 10 min at 4°C.
9. Transfer supernatant to a new tube. (SAFE STOP) 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 IV). This 50 µl sample may be stored at -20°C overnight.

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

1. To the 50 µl chromatin sample (from Step 9 in Section III), add 100 µl nuclease-free water, 6 µl 5 M NaCl #7010, and 2 µl RNAse A #7013. Vortex to mix and incubate samples at 37°C for 30 min.
2. To each RNAse A-digested sample, add 2 µl Proteinase K. Vortex to mix and incubate samples at 65°C for 2 h.
3. Purify DNA from samples using DNA purification spin columns as described in Section VII. (SAFE STOP) DNA may be stored at -20°C for up to 6 months.
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 nuclease-free water 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 B.

V. Chromatin Immunoprecipitation

For optimal ChIP results, use approximately 5 to 10 µg of digested, cross-linked chromatin (as determined in Section IV) per immunoprecipitation. This should be roughly equivalent to a single 100 µl IP prep from 25 mg of disaggregated tissue or 4 x 10⁶ tissue culture cells. Typically, 100 µl of digested chromatin is diluted into 400 µl 1X ChIP Buffer prior to the addition of antibodies. However, if more than 100 µl of chromatin is required per IP, 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.

Before starting

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

• Remove and warm 200X Protease Inhibitor Cocktail (PIC) #7012. Make sure PIC is completely thawed.
• Remove and warm 10X ChIP Buffer #7008 and ensure SDS is completely in solution.
• Thaw digested chromatin preparation (from Step 9 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. Store at room temperature until use.
• 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. Store at room temperature until use.

1. In one tube, prepare enough 1X ChIP Buffer for the dilution of digested chromatin into the desired number of immunoprecipitations: 400 µl of 1X ChIP Buffer (40 µl of 10X ChIP Buffer + 360 µl water) + 2 µl 200X PIC per immunoprecipitation. When determining the number of immunoprecipitations, remember to include the positive control Histone H3 (D2B12) XP^® Rabbit mAb #4620 and negative control Normal Rabbit IgG antibody #2729 samples. Place mix on ice.
2. To the prepared 1X ChIP Buffer, add the equivalent of 100 µl (5 to 10 µg of chromatin) of the digested, cross-linked chromatin preparation (from Step 9 in Section III) per 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 200X PIC + 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 VI).
4. For each immunoprecipitation, transfer 500 µl of the diluted chromatin 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) XP^® Rabbit mAb #4620, add 10 µl to the IP sample. For the negative control Normal Rabbit IgG #2729, add 1 µl (1 µg) to 2 µl (2 µg) to the IP sample. If using antibodies from Cell Signaling Technology, please see recommended dilution listed on the datasheet or product webpage and calculate the amount (µg) of IgG antibody for negative control based on the Cell Signaling Antibody concentration for a fair comparison. Incubate IP samples 4 h to overnight at 4°C with rotation.

   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.

5. Resuspend ChIP-Grade Protein G Magnetic Beads #9006 by gently vortexing. Immediately add 30 µl of Protein G Magnetic Beads 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 in a magnetic separation rack #7017. 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. Add 1 ml of high salt wash to the beads and incubate at 4°C for 5 min with rotation.
9. Pellet protein G magnetic beads in each immunoprecipitation by placing the tubes in 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.

• Remove and 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, elutions can be performed at room temperature with rotation, but may not be as complete.
4. Pellet protein G magnetic beads by placing the tubes in a magnetic separation rack and wait 1 to 2 min for solution to clear.
5. Carefully transfer eluted chromatin supernatant to a new tube.
6. To all tubes, including the 2% input sample from Step 1, reverse cross-links by adding 6 µl 5M NaCl and 2 µl Proteinase K #10012, and incubate 2 h at 65°C. This incubation can be extended overnight.
7. 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 to room temperature before adding DNA Binding Buffer #10007 (Section VII, Step 1).

VII. DNA Purification Using Spin Columns

Before starting

• (!!) Add 24 ml of ethanol (96-100%) to 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 collection tube #10010 for each DNA sample from Section V.

1. Add 750 µl DNA Binding Buffer #10007 to each DNA sample and vortex briefly.
   • 5 volumes of DNA Binding Buffer should be used for every 1 volume of sample.
2. Transfer 450 µl of each sample from Step 1 to a DNA spin column in collection tube.
3. Centrifuge at 18,500 x g in a microcentrifuge for 30 sec.
4. Remove the spin column from the collection tube and discard the liquid. Replace spin column in the collection tube.
5. Transfer the remaining 450 µl of each sample from Step 1 to the spin column in collection tube. Repeat Steps 3 and 4.
6. Add 750 µl of DNA Wash Buffer #10008 to the spin column in 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. Replace spin column in the collection tube.
9. Centrifuge at 18,500 x g in a microcentrifuge for 30 sec.
10. Discard collection tube and liquid. Retain spin column.
11. Add 50 µl of DNA Elution Buffer #10009 to each spin column and place into a clean 1.5 ml microcentrifuge tube.
12. Centrifuge at 18,000 x g in a microcentrifuge for 30 sec to elute DNA.
13. Remove and discard DNA spin column. Eluate is now purified DNA. (SAFE STOP) Samples can be stored at -20°C.

VIII. 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 (#7014 + #7015) 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 nonspecific PCR products.
• PCR primer selection is critical. Primers should be designed with close adherence to the following criteria:

Standard PCR Method

1. Label the appropriate number of 0.2 ml PCR tubes for the number of samples to be analyzed. These should include the 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.

4. Start the following PCR reaction program:

5. Remove 10 µl of each PCR product for analysis by 2% agarose gel or 10% polyacrylamide gel electrophoresis with a 100 bp DNA marker. The expected size of the PCR product 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 plate with aluminum foil to avoid light and store at 4°C up to 4 hours or -20°C overnight until machine is ready for use.

4. Start the following PCR reaction program:

5. Analyze quantitative PCR results using the software provided with the real-time PCR machine. Alternatively, one can calculate the IP efficiency manually using the Percent Input Method and the equation shown below. With this method, signals obtained from each immunoprecipitation are expressed as a percent of the total input chromatin.

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

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

IX. NG-Sequencing Library Construction

The immuno-enriched DNA samples prepared with this kit are directly compatible with ChIP-seq. For downstream NG-sequencing DNA library construction, use a DNA library preparation protocol or kit compatible with your downstream sequencing platform. For sequencing on Illumina^® platforms, we recommend SimpleChIP^® ChIP-seq DNA Library Prep Kit for Illumina^® #56795 and its associated index primers SimpleChIP^® ChIP-seq Multiplex Oligos for Illumina^® (Single Index Primers) #29580 or SimpleChIP^® ChIP-seq Multiplex Oligos for Illumina^® (Dual Index Primers) #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 an Agilent High Sensitivity DNA Kit (Agilent Technologies, Cat# G2938-90322), 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 2-10 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 to the right provides a range for the expected yield of chromatin from 25 mg of tissue compared to 4 x 10⁶ HeLa cells, and the expected DNA concentration, as determined in Section IV of the protocol. For each tissue type, disaggregation using a Medimachine (BD Biosciences) or a Dounce homogenizer yielded similar amounts of chromatin. However, chromatin processed from tissues disaggregated using the Medimachine typically gave higher IP efficiencies than chromatin processed from tissues disaggregated using a Dounce homogenizer. A Dounce homogenizer is strongly recommended for disaggregation of brain tissue, as the Medimachine does not adequately disaggregate brain tissue into a single-cell suspension. For optimal ChIP results, we recommend using 5 to 10 µg of digested, cross-linked chromatin per immunoprecipitation; therefore, some tissues may require harvesting more than 25 mg per each immunoprecipitation.

APPENDIX B: 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 tissue or number of cells used in the digest. Below is a protocol for determination of the optimal digestion conditions for a specific tissue or cell type.

1. Prepare cross-linked nuclei from 125 mg of tissue or 2 X 10⁷ cells (equivalent of 5 IP preps), as described in Sections I, II, and III. Stop after Step 2 of Section III 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 stock 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 digest by adding 10 µl of 0.5 M EDTA and placing tubes on ice.
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 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-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.
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 h.
13. Remove 20 µl of each sample and determine DNA fragment size by electrophoresis on a 1% agarose gel with a 100 bp 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 10 times the volume of Micrococcal Nuclease stock that should be added to one immunoprecipitation preparation (25 mg of disaggregated tissue cells or 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 stock Micrococcal Nuclease should be added to one IP prep during the digestion of chromatin in Section III.
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. Alternatively, the digestion time can be changed to increase or decrease the extent of DNA fragmentation.

APPENDIX C: Troubleshooting Guide

Chromatin IP

Specific for product: SimpleChIP^® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005.

Required Reagents

Reagents Included:

1. Glycine Solution (10X) #7005
2. Buffer A (4X) #7006
3. Buffer B (4X) #7007
4. ChIP Buffer (10X) #7008
5. ChIP Elution Buffer (2X) #7009
6. 5 M NaCl #7010
7. 0.5 M EDTA #7011
8. ChIP-Grade Protein G Magnetic Beads #9006
9. DNA Binding Buffer #10007
10. DNA Wash Buffer (add 4x volume ethanol before use) #10008
11. DNA Elution Buffer #10009
12. DNA Purification Columns #10010
13. Protease Inhibitor Cocktail (200X) #7012
14. RNAse A (10 mg/ml) #7013
15. Micrococcal Nuclease (2000 gel units/µl) #10011
16. Proteinase K (20 mg/ml) #10012
17. SimpleChIP^® Human RPL30 Exon 3 Primers 1 #7014
18. SimpleChIP^® Mouse RPL30 Intron 2 Primers 1 #7015
19. Histone H3 (D2B12) XP^® Rabbit mAb (ChIP Formulated) #4620
20. Normal Rabbit IgG #2729
21. DTT (Dithiothreitol) #7016

Reagents Not Included:

1. Magnetic Separation Rack #7017 / 14654
2. Phosphate Buffered Saline (PBS-1X) pH7.2 (Sterile) #9872
3. Nuclease-free Water #12931
4. Ethanol (96-100%)
5. Formaldehyde (37% Stock)
6. Taq DNA Polymerase
7. dNTP Mix

I. Tissue Cross-linking and Sample Preparation

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

Before starting:

• Remove and warm 200X Protease Inhibitor Cocktail (PIC) and 10X Glycine Solution. Make sure PIC is completely thawed.
• Prepare 3 ml of Phosphate Buffered Saline (PBS) + 15 µl 200X PIC per 25 mg of tissue to be processed and place on ice.
• Prepare 45 µl of 37% formaldehyde per 25 mg of tissue to be processed and keep at room temperature. Use fresh formaldehyde that is not past the manufacturer's expiration date.

A. Cross-linking

1. Weigh the fresh or frozen tissue sample. Use 25 mg of tissue for each IP to be performed.
2. Place tissue sample in a 60 mm or 100 mm dish and finely mince using a clean scalpel or razor blade. Keep 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.
4. Add 1 ml of PBS + PIC per 25 mg tissue to the conical tube.
5. To crosslink proteins to DNA, add 45 µl of 37% formaldehyde per 1 ml of PBS + PIC and rock at room temp for 20 min. Final formaldehyde concentration is 1.5%.
6. Stop cross-linking by adding 100 µl of 10X Glycine per 1 ml of PBS + PIC and mix for 5 min at room temperature.
7. Centrifuge tissue at 1,500 rpm in a benchtop centrifuge for 5 min at 4°C.
8. Remove supernatant and wash one time with 1 ml PBS + PIC per 25 mg tissue.
9. Repeat centrifugation at 1,500 rpm in a benchtop centrifuge for 5 min at 4°C.
10. Remove supernatant and resuspend tissue in 1 ml PBS + PIC per 25 mg tissue and store on ice. Disaggregate tissue into single-cell suspension using a Medimachine (Part B) or Dounce homogenizer (Part C).

B. Tissue Disaggregation Using Medimachine from BD Biosciences (part #340587)

1. Cut off the end of a 1000 µL pipette tip to enlarge the opening for transfer of tissue chunks.
2. Transfer 1 ml of tissue resuspended in PBS + PIC into the top chamber of a 50 mm medicone (part #340592).
3. Grind tissue for 2 min according to manufacturer's instructions.
4. Collect cell suspension from the bottom chamber of the medicone using a 1 ml syringe and 18 gauge blunt needle. Transfer cell suspension to a 15 ml conical tube and place on ice.
5. Repeat steps 2 to 4 until all the tissue is processed into a homogenous suspension.
6. If more grinding is necessary, add more PBS + PIC to tissue. Repeat steps 2 to 5 until all tissue is ground into a homogeneous suspension.
7. Check for single-cell suspension by microscope (optional).
8. Centrifuge cells at 1,500 rpm in a bench top centrifuge for 5 min at 4°C.
9. Remove supernatant from cells and immediately continue with Nuclei Preparation and Chromatin Digestion (Section III).

C. Tissue Disaggregation Using a Dounce Homogenizer

1. Transfer tissue resuspended in PBS + PIC to a Dounce homogenizer.
2. Disaggregate tissue pieces with 20-25 strokes. Check for single-cell suspension by microscope (optional).
3. Transfer cell suspension to a 15 ml conical tube and centrifuge at 1,500 rpm in a benchtop centrifuge for 5 min at 4°C.
4. Remove supernatant from cells and immediately continue with Nuclei Preparation and Chromatin Digestion (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 HeLa cells, this 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 IV). Include one extra dish of cells in experiment to be used for determination of cell number using a hemocytometer.

Before starting

• Remove and warm 200X Protease Inhibitor Cocktail (PIC) #7012 and 10X Glycine Solution #7005. Make sure PIC is completely thawed.
• Prepare 2 ml of Phosphate Buffered Saline (PBS) + 10 µl 200X PIC per 15 cm dish to be processed and place on ice.
• Prepare 40 ml of PBS per 15 cm dish to be processed and place on ice.
• Prepare 540 µl of 37% formaldehyde per 15 cm dish of 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 540 µl of 37% formaldehyde to each 15 cm culture dish containing 20 ml medium. Swirl briefly to mix and incubate 10 min at room temperature. Final formaldehyde concentration is 1%. Addition of formaldehyde may result in a color change of the medium.
2. Add 2 ml of 10X glycine to each 15 cm dish containing 20 ml medium, swirl briefly to mix, and incubate 5 min 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 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 III).
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.
5. Add 2 ml ice-cold PBS + PIC to each 15 cm dish. Scrape cells into cold buffer. Combine cells from all culture dishes into one 15 ml conical tube.
6. Centrifuge cells at 1,500 rpm in a benchtop centrifuge for 5 min at 4°C. Remove supernatant and immediately continue with Nuclei Preparation and Chromatin Digestion (Section III).

III. Nuclei Preparation and Chromatin Digestion

One immunoprecipitation preparation (IP prep) is defined as 25 mg of disaggregated tissue or 4 x 10⁶ tissue culture cells.

Before starting

• Remove and warm 200X Protease Inhibitor Cocktail (PIC) #7012. 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.

  IMPORTANT: Once in solution, store 1M DTT at -20°C.

• Remove and 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 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 per IP prep and place on ice.

1. Resuspend cells in 1 ml ice-cold 1X Buffer A + DTT + PIC per IP prep. Incubate on ice for 10 min. Mix by inverting tube every 3 min.
2. Pellet nuclei by centrifugation at 3,000 rpm in a benchtop centrifuge for 5 min at 4°C. Remove supernatant and resuspend pellet in 1 ml ice-cold 1X Buffer B + DTT per IP prep. Repeat centrifugation, remove supernatant, and resuspend pellet in 100 µl 1X Buffer B +DTT per IP prep. Transfer sample to a 1.5 ml microcentrifuge tube, up to 1 ml total per tube.
3. 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 frequent mixing to digest DNA to length of approximately 150-900 bp. Mix by inversion every 3 to 5 min. The amount of Micrococcal Nuclease required to digest DNA to the optimal length may need to be determined empirically for individual tissues and cell lines (see Appendix B). HeLa nuclei digested with 0.5 µl Micrococcal Nuclease per 4 x 10⁶ cells and mouse liver tissue digested with 0.5 µl Micrococcal Nuclease per 25 mg of tissue gave the appropriate length DNA fragments.
4. Stop digest by adding 10 µl of 0.5 M EDTA #7011 per IP prep and placing tube on ice.
5. Pellet nuclei by centrifugation at 13,000 rpm in a microcentrifuge for 1 min at 4°C and remove supernatant.
6. Resuspend nuclear pellet in 100 µl of 1X ChIP Buffer + PIC per IP prep and incubate on ice for 10 min.
7. Sonicate up to 500 µl of lysate per 1.5 ml microcentrifuge tube with several pulses to break nuclear membrane. Incubate samples for 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-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.
8. Clarify lysates by centrifugation at 10,000 rpm in a microcentrifuge for 10 min at 4°C.
9. 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 IV).

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

1. To the 50 µl chromatin sample (from Step 9 in Section III), add 100 µl nuclease-free water, 6 µl 5 M NaCl #7010, and 2 µl RNAse A #7013. Vortex to mix and incubate samples at 37°C for 30 min.
2. To each RNAse A-digested sample, add 2 µl Proteinase K. Vortex to mix and incubate samples at 65°C for 2 h.
3. Purify DNA from samples using DNA purification spin columns as described in Section VII.
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 nuclease-free water 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 B.

V. Chromatin Immunoprecipitation

For optimal ChIP results, use approximately 5 to 10 µg of digested, cross-linked chromatin (as determined in Section IV) per immunoprecipitation. This should be roughly equivalent to a single 100 µl IP prep from 25 mg of disaggregated tissue or 4 x 10⁶ tissue culture cells. Typically, 100 µl of digested chromatin is diluted into 400 µl 1X ChIP Buffer prior to the addition of antibodies. However, if more than 100 µl of chromatin is required per IP, 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.

Before starting

• Remove and warm 200X Protease Inhibitor Cocktail (PIC) #7012. Make sure PIC is completely thawed.
• Remove and warm 10X ChIP Buffer #7008 and ensure SDS is completely in solution.
• Thaw digested chromatin preparation (from Step 9 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. Store at room temperature until use.
• 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. Store at room temperature until use.

1. In one tube, prepare enough 1X ChIP Buffer for the dilution of digested chromatin into the desired number of immunoprecipitations: 400 µl of 1X ChIP Buffer (40 µl of 10X ChIP Buffer + 360 µl water) + 2 µl 200X PIC per immunoprecipitation. When determining the number of immunoprecipitations, remember to include the positive control Histone H3 (D2B12) XP^® Rabbit mAb #4620 and negative control Normal Rabbit IgG antibody #2729 samples. Place mix on ice.
2. To the prepared 1X ChIP Buffer, add the equivalent of 100 µl (5 to 10 µg of chromatin) of the digested, cross-linked chromatin preparation (from Step 9 in Section III) per 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 200X PIC + 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 VI).
4. For each immunoprecipitation, transfer 500 µl of the diluted chromatin 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) XP^® Rabbit mAb #4620, add 10 µl to the IP sample. For the negative control Normal Rabbit IgG #2729, add 1 µl (1 µg) to 2 µl (2 µg) to the IP sample. Incubate IP samples 4 h to overnight at 4°C with rotation.

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.

5. Resuspend ChIP-Grade Protein G Magnetic Beads #9006 by gently vortexing. Immediately add 30 µl of Protein G Magnetic Beads 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 in a magnetic separation rack #7017. 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. Add 1 ml of high salt wash to the beads and incubate at 4°C for 5 min with rotation.
9. Pellet protein G magnetic beads in each immunoprecipitation by placing the tubes in 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

• Remove and 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, elutions can be performed at room temperature with rotation, but may not be as complete.
4. Pellet protein G magnetic beads by placing the tubes in a magnetic separation rack and wait 1 to 2 min for solution to clear.
5. Carefully transfer eluted chromatin supernatant to a new tube.
6. To all tubes, including the 2% input sample from Step 1, reverse cross-links by adding 6 µl 5M NaCl and 2 µl Proteinase K #10012, and incubate 2 h at 65°C. This incubation can be extended overnight.
7. Immediately proceed to Section VII. Alternatively, samples can be stored at -20°C. However, to avoid formation of a precipitate, be sure to warm samples to room temperature before adding DNA Binding Buffer #10007 (Section VII, Step 1).

VII. DNA Purification Using Spin Columns

Before starting

• Add 24 ml of ethanol (96-100%) to 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 collection tube #10010 for each DNA sample from Section V.

1. Add 750 µl DNA Binding Buffer #10007 to each DNA sample and vortex briefly.
   • 5 volumes of DNA Binding Buffer should be used for every 1 volume of sample.
2. Transfer 450 µl of each sample from Step 1 to a DNA 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 450 µl of each sample from Step 1 to the spin column in collection tube. Repeat Steps 3 and 4.
6. Add 750 µl of DNA Wash Buffer #10008 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 Buffer #10009 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 spin column. Eluate is now purified DNA. Samples can be stored at -20°C.

VIII. 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 (#7014 + #7015) 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 nonspecific PCR products.
• PCR primer selection is critical. Primers should be designed with close adherence to the following criteria:

Standard PCR Method

1. Label the appropriate number of 0.2 ml PCR tubes for the number of samples to be analyzed. These should include the 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.

4. Start the following PCR reaction program:

5. Remove 10 µl of each PCR product for analysis by 2% agarose gel or 10% polyacrylamide gel electrophoresis with a 100 bp DNA marker. The expected size of the PCR product 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.

4. Start the following PCR reaction program:

5. Analyze quantitative PCR results using the software provided with the real-time PCR machine. Alternatively, one can calculate the IP efficiency manually using the Percent Input Method and the equation shown below. With this method, signals obtained from each immunoprecipitation are expressed as a percent of the total input chromatin.

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

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

IX. NG-Sequencing Library Construction

The immuno-enriched DNA samples prepared with this kit are directly compatible with ChIP-seq. For downstream NG-sequencing DNA library construction, use a DNA library preparation protocol or kit compatible with your downstream sequencing platform. For sequencing on Illumina^® platforms, we recommend NEBNext^® Ultra^™ II Library Prep Kit for Illumina^® (New England Biolabs; Cat #E7645S/L).

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 an Agilent High Sensitivity DNA Kit (Agilent Technologies, Cat# G2938-90322), 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 2-10 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 to the right provides a range for the expected yield of chromatin from 25 mg of tissue compared to 4 x 10⁶ HeLa cells, and the expected DNA concentration, as determined in Section IV of the protocol. For each tissue type, disaggregation using a Medimachine (BD Biosciences) or a Dounce homogenizer yielded similar amounts of chromatin. However, chromatin processed from tissues disaggregated using the Medimachine typically gave higher IP efficiencies than chromatin processed from tissues disaggregated using a Dounce homogenizer. A Dounce homogenizer is strongly recommended for disaggregation of brain tissue, as the Medimachine does not adequately disaggregate brain tissue into a single-cell suspension. For optimal ChIP results, we recommend using 5 to 10 µg of digested, cross-linked chromatin per immunoprecipitation; therefore, some tissues may require harvesting more than 25 mg per each immunoprecipitation.

APPENDIX B: 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 tissue or number of cells used in the digest. Below is a protocol for determination of the optimal digestion conditions for a specific tissue or cell type.

1. Prepare cross-linked nuclei from 125 mg of tissue or 2 X 10⁷ cells (equivalent of 5 IP preps), as described in Sections I, II, and III. Stop after Step 2 of Section III 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 stock 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 digest by adding 10 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. 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-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 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 h.
13. Remove 20 µl of each sample and determine DNA fragment size by electrophoresis on a 1% agarose gel with a 100 bp 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 10 times the volume of Micrococcal Nuclease stock that should be added to one immunoprecipitation preparation (25 mg of disaggregated tissue cells or 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 stock Micrococcal Nuclease should be added to one IHC prep during the digestion of chromatin in Section III.
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. Alternatively, the digestion time can be changed to increase or decrease the extent of DNA fragmentation.

APPENDIX C: Troubleshooting Guide

CUT&RUN Protocol

I. Cell Preparation and Binding of Primary Antibody

NOTE: Steps for cell preparation (step 6-16) should be performed in succession at room temperature to minimize stress on the cells. To minimize DNA fragmentation, avoid vigorous vortexing and cavitation during resuspension.

NOTE: This protocol is written for the use of 100,000 cells per reaction. However, these same reaction conditions can be used for 10,000 to 250,000 cells per sample.

NOTE: The amount of digitonin recommended for cell permeabilization is in excess and should be sufficient for permeabilization of most cell lines. However, not all cell lines exhibit the same sensitivity to digitonin. Before you begin your experiment, it is recommended that you test your specific cell line by following the protocol provided in Appendix A. Digitonin treatment should result in permeabilization of >90% of the cell population.

Before Starting:

! All buffer volumes should be increased proportionally based on the number of CUT&RUN reactions being performed.

• Remove and warm 200X Protease Inhibitor Cocktail and 100X Spermidine. Make sure both are completely thawed.
• Remove and warm Digitonin Solution at 90-100°C for 5 min and make sure it is completely thawed and in solution. Immediately place the thawed Digitonin Solution on ice.

  NOTE: Digitonin Solution should be stored at -20°C. Please keep on ice during use and store at -20°C when finished for the day.

• Prepare 1X Wash Buffer (2 ml for each cell line and additional 100 µl for each reaction or input sample). For example, to prepare 2.5 ml of 1X Wash Buffer, add 250 µl 10X Wash Buffer + 25 µl 100X Spermidine + 12.5 µl 200X Protease Inhibitor Cocktail + 2212.5 µl water. Equilibrate it to room temperature to minimize stress on the cells.
• For each reaction, prepare 1 µl 100X Spermidine + 0.5 µl 200X Protease Inhibitor Cocktail + 2.5 µl Digitonin Solution + 96 µl Antibody Binding Buffer and place on ice (100 µl per reaction).
• Place Concanavalin A Bead Activation Buffer on ice.

1. Carefully resuspend Concanavalin A Magnetic Beads by gently pipetting up and down, making sure not to splash any bead suspension out of the tube. Transfer 10 µl of the bead suspension per each CUT&RUN reaction to a new 1.5 ml microcentrifuge tube.

NOTE: Avoid vortexing of the Concanavalin A Magnetic Bead suspension as repeated vortexing may displace the Concanavalin A from the beads.

2. Add 100 µl Concanavalin A Bead Activation Buffer per 10 µl beads. Gently mix beads by pipetting up and down.
3. Place tube on a magnetic rack until solution becomes clear (30 sec to 2 min) and then remove the liquid.

   NOTE: To avoid loss of beads, remove liquid using a pipetman. Do not aspirate using a vacuum.

4. Remove tubes from the magnetic rack. Wash the beads a second time by repeating steps 2 and 3.
5. Add a volume of Concanavalin A Bead Activation Buffer equal to the initial volume of bead suspension added (10 µl per sample) and resuspend by pipetting up and down. Store the activated beads on ice until Section I, Step 13.
6. Harvest fresh cell cultures at room temperature to minimize stress on the cells. Collect 100,000 cells for each antibody/MNase reaction and an additional 100,000 cells for the input sample. Be sure to include reactions for the positive control Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb and the negative control Rabbit (DA1E) mAb IgG XP^® Isotype Control (CUT&RUN).
7. Centrifuge cell suspension for 3 min at 600 x g at room temperature and remove the liquid.
8. Resuspend cell pellet in 1 ml of 1X Wash Buffer (+ Spermidine + PIC) at room temperature by gently pipetting up and down.
9. Centrifuge for 3 min at 600 x g at room temperature and remove the liquid.
10. Wash the cell pellet a second time by repeating steps 8 and 9 one time.
11. For each 100,000 cells, add 100 µl of 1X Wash Buffer (+ Spermidine + PIC) and resuspend the cell pellet by gently pipetting up and down.
12. Transfer 100 µl of cells to a new tube and store at 4°C until Section IV. This is your input sample.

    NOTE: The input sample will be incubated at 55°C later in the protocol, so it is recommended to use a safe-lock 1.5 ml tube to reduce evaporation during the incubation.

13. Resuspend the activated Concanavalin A Magnetic Beads (from Step 5) by pipetting up and down. Add 10 µl of activated bead suspension per 100,000 cells to the washed cell suspension in Step 11.
14. Rotate the sample for 5 min at room temperature.

    NOTE: Concanavalin A Magnetic Beads may clump or stick to the sides of the tube. Beads can be resuspended by pipetting up and down.

15. Briefly centrifuge the sample at 100 x g to remove cell:bead suspension from the cap of the tube. Place the tube on the magnetic rack until the solution turns clear (30 sec to 2 min), then remove and discard the liquid.
16. Remove tube from the stand. Add 100 µl of Antibody Binding Buffer (+ Spermidine + PIC + digitonin) per 100,000 cells and place on ice.
17. Aliquot 100 µl of the cell:bead suspension into a separate 1.5 ml tube for each reaction and place on ice.
18. Add the appropriate amount of antibody to each reaction and mix gently by pipetting up and down.

    NOTE: The amount of antibody required for CUT&RUN varies and should be determined by the user. For the positive control Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb, add 2 µl of antibody to the sample. For the negative control Rabbit (DA1E) mAb IgG XP^® Isotype Control (CUT&RUN) #66362, add 5 µl to the sample. We strongly recommend using the negative control antibody and NOT a no-antibody control, because the latter results in high levels of non-specific MNase digestion and high background signal. We recommend using the input sample for comparison with both qPCR and NG-seq analysis.

19. Rotate tubes at 4°C for 2 hr. This step can be extended to overnight.

II. Binding of pAG-MNase Enzyme

Before Starting:

! All buffer volumes should be increased proportionally based on the number of CUT&RUN reactions being performed.

• Remove and warm Digitonin Solution at 90-100°C for 5 min and make sure it is completely thawed and in solution. Immediately place the thawed Digitonin Solution on ice.

NOTE: Digitonin Solution should be stored at -20°C. Please keep on ice during use and store at -20°C when finished for the day.

• For each reaction, prepare 1.05 ml of Digitonin Buffer (105 µl 10X Wash Buffer + 10.5 µl 100X Spermidine + 5.25 µl 200X Protease Inhibitor Cocktail + 26.25 µl Digitonin Solution + 903 µl water).
• For each reaction, make a pAG-MNase pre-mix by adding 50 µl of Digitonin Buffer (described above) and 1.5 µl of pAG-MNase Enzyme to a new tube. For example, for 10 reactions, transfer 500 µl of Digitonin Buffer to a new tube and add 15 µl of pAG-MNase Enzyme. Mix by gently pipetting up and down and place on ice.

1. Briefly centrifuge samples from Section I, Step 19 at 100 x g to remove cell:bead suspension from the caps of the tubes.
2. Place the tubes on the magnetic rack until the solution turns clear (30 sec to 2 min) and then remove the liquid.
3. Remove tubes from the magnetic rack and add 1 ml of Digitonin Buffer (+ Spermidine + PIC + digitonin). Resuspend beads by gently pipetting up and down.
4. Place the tubes on the magnetic rack until the solution turns clear (30 sec to 2 min) and then remove the liquid.
5. Remove tubes from magnetic rack. Add 50 µl of pAG-MNase pre-mix to each tube and gently mix the sample by pipetting up and down.
6. Rotate tubes at 4°C for 1 hr.

III. DNA Digestion and Diffusion

Before Starting:

! All buffer volumes should be increased proportionally based on the number of CUT&RUN reactions being performed.

• Remove and warm Digitonin Solution at 90-100°C for 5 min and make sure it is completely thawed and in solution. Immediately place the thawed Digitonin Solution on ice.

  NOTE: Digitonin Solution should be stored at -20°C. Please keep on ice during use and store at -20°C when finished for the day.

• For each reaction, prepare 2.15 ml Digitonin Buffer (215 µl 10X Wash Buffer + 21.5 µl 100X Spermidine + 10.75 µl 200X Protease Inhibitor Cocktail + 53.75 µl Digitonin Solution + 1.849 ml water).
• For each reaction, prepare 150 µl of 1X Stop Buffer (37.5 µl 4X Stop Buffer + 3.75 µl Digitonin Solution + 0.75 µl RNAse A + 108 µl water).
• Place Calcium Chloride on ice.
• Optional: Sample Normalization Spike-In DNA can be added into the 1X Stop Buffer if sample normalization is desired (for example, see Figure 8 in Section VI). For qPCR analysis, we recommend adding 5 µl (5 ng) of Spike-In DNA to each reaction. For NG-seq analysis, we recommend diluting the Sample Normalization Spike-In DNA 500-fold into nuclease-free water and then adding 5 µl (10 pg) of Spike-In DNA to each reaction. This will result in at least a thousand normalization reads per million total sequencing reads if you use the entire sample for sequencing. If starting with more or less than 100,000 cells per reaction, proportionally scale the volume of Sample Normalization Spike-In DNA for NG-seq analysis. For example, for 25,000 starting cells, add 1.25 µl (2.5 pg) of 1:500 diluted Spike-In DNA to each reaction.

1. Briefly centrifuge samples from Section II, Step 6 at 100 x g to remove cell:bead suspension from the caps of the tubes.
2. Place the tubes on the magnetic separation rack until the solution turns clear (30 sec to 2 min) and then remove the liquid.
3. Remove tubes from the magnetic separation rack. Add 1 ml of Digitonin Buffer (+ Spermidine + PIC + digitonin) and resuspend beads by gently pipetting up and down.
4. Repeat steps 2 and 3 one time.
5. Place the tubes on the magnetic rack until the solution turns clear (30 sec to 2 min) and then remove the liquid.
6. Remove tubes from magnetic rack. Add 150 µl of Digitonin Buffer (+ Spermidine + PIC + digitonin) to each tube and mix by pipetting up and down.
7. Place tubes on ice for 5 min to cool before digestion.
8. Activate MNase by adding 3 µl cold Calcium Chloride to each tube and mix by pipetting up and down.
9. Incubate samples at 4°C for 30 min.

   NOTE: Digestion should be performed in a 4°C cooling block or refrigerator. The temperature of ice can get as low as 0°C, which can limit digestion and decrease signal.

10. Add 150 µl of 1X Stop Buffer (+ digitonin + RNAse A + spike-in DNA [optional]) to each sample and mix by pipetting up and down.
11. Incubate the tubes at 37°C for 10 min without shaking to release DNA fragments into the solution.

    NOTE: This incubation step can be increased to 30 min.

12. Centrifuge at 4°C for 2 min at 16,000x g and place the tubes on a magnetic rack until the solution is clear (30 sec to 2 min).
13. Transfer the supernatant to a new 1.5 ml microcentrifuge tube and place on ice. This is your enriched chromatin sample.
14. Immediately proceed to Section V. (SAFE STOP) Alternatively, samples can be stored at -20°C for up to 1 week. However, be sure to warm samples to room temperature before DNA purification (Section V).

IV. Preparation of the Input Sample

! All buffer volumes should be increased proportionally based on the number of input samples being prepared.

Before Starting:

• Remove and warm DNA Extraction Buffer. Make sure it is completely thawed.
• For each input sample, prepare 2 µl Proteinase K + 0.5 µl RNAse A + 197.5 µl DNA Extraction Buffer (200 µl per input sample).

1. Add 200 µl of DNA Extraction Buffer (+ Proteinase K + RNAse A) to the 100 µl cell suspension from Section I step 12. Mix by pipetting up and down.
2. Place the tube at 55°C for 1 hr with shaking.
3. Place the tubes on ice for 5 min to completely cool the sample.
4. Lyse the cells and fragment the chromatin by sonicating the input samples. Incubate samples on ice for 30 seconds between pulses.

   NOTE: Sonication conditions may need to be determined empirically by testing different sonicator power settings and/or durations of sonication, following the protocol in Appendix B. Optimal sonication conditions will generate chromatin fragments ranging in size from 100-600 bp. Sonication for 5 sets of 15-sec pulses using a VirTis Virsonic 100 Ultrasonic Homogenizer/Sonicator at setting 6 with a 1/8-inch probe sufficiently fragments the input chromatin.

5. Clarify lysates by centrifugation at 18,500 x g for 10 min at 4°C. Transfer supernatant to a new 1.5 ml microcentrifuge tube.
6. Immediately proceed to Section V DNA Purification. (SAFE STOP) Alternatively, samples can be stored at -20°C for up to 1 week. However, be sure to warm samples to room temperature before DNA purification procedures (Section V).

V. DNA Purification

DNA can be purified from input and enriched chromatin samples using DNA spin columns, as described in Section A, or phenol/chloroform extraction followed by ethanol precipitation as described in Section B. Purification using DNA spin columns is simple and fast, providing good recovery of DNA fragments above 35 bp (Figure 7A, Lane 2). Phenol/chloroform extraction followed by ethanol precipitation is more difficult, but provides better recovery of DNA fragments below 35 bp (Figure 7A, Lane 3); however, as shown in Figure 7B, the majority of DNA fragments generated in the CUT&RUN assay are larger than 35 bp. Therefore, DNA spin columns provide a quick and simple method for purification of > 98% of the total CUT&RUN DNA fragments.

Purified DNA can be quantified prior to NG-seq analysis using a picogreen-based DNA quantification assay. For CUT&RUN reactions containing 100,000 cells, the expected DNA yield for a CUT&RUN reaction ranges from 0.5 to 10 ng per reaction for transcription factors and cofactors, and 1 to 20 ng per reaction for histone modifications.

FIGURE 7. Comparison of DNA purification using spin columns or phenol/chloroform extraction followed by ethanol precipitation. (A) A low range DNA ladder mix (lane 1, unpurified) was purified using either DNA Purification Buffers and Spin Columns (ChIP, CUT&RUN) #14209 (lane 2) or phenol/chloroform extraction followed by ethanol precipitation (lane 3) and separated by electrophoresis on a 4% agarose gel. As shown, phenol/chloroform followed by ethanol precipitation efficiently recovers all DNA fragment sizes, while DNA spin columns recover DNA fragments ≥ 35 bp. (B) DNA was purified using phenol/chloroform extraction followed by ethanol precipitation from a CUT&RUN assay performed using TCF4/TCF7L2 (C48H11) Rabbit mAb #2569. The size of the DNA fragments in the library was analyzed using a Bioanalyzer (Agilent Technologies). The adaptor and barcode sequences added to the library during construction account for 140 bp in fragment length. Therefore, starting 35 bp DNA fragments would be 175 bp in length after library preparation (indicated with blue vertical line in figure). As shown, less than 2% of the total CUT&RUN enriched DNA fragments are less than 175 bp (starting length of 35 bp), suggesting that DNA purification spin columns are sufficient for capture of > 98% of the total CUT&RUN DNA fragments.

A. DNA Purification Using Spin Columns

NOTE: DNA can be purified from input and enriched chromatin samples using the Cell Signaling^® DNA Purification Buffers and Spin Columns (ChIP, CUT&RUN) #14209 (not included in this kit) and the modified protocol below. Steps 1 through 5 are modified to reflect the requirement for adding 5 volumes (1.5 ml) of DNA Binding Buffer to the 300 µl of input and enriched chromatin samples.

Before starting:

• !! Add 24 ml of ethanol (96-100%) to 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 collection tube #10010 for each enriched chromatin sample or input sample to be purified.

1. Add 1.5 ml DNA Binding Buffer to each input and enriched chromatin sample and vortex briefly.

   NOTE: 5 volumes of DNA Binding Buffer should be used for every 1 volume of sample.

2. Transfer 600 µl of each sample from Step 1 to a DNA spin column in collection tube.
3. Centrifuge at 18,500 x g in a microcentrifuge for 30 sec.
4. Remove the spin column from the collection tube and discard the liquid. Replace the spin column in the empty collection tube.
5. Repeat steps 2-4 until the entire sample from Step 1 has been spun through the spin column. Replace the spin column in the empty collection tube.
6. Add 750 µl of DNA Wash Buffer to the spin column in collection tube.
7. Centrifuge at 18,500 x g in a microcentrifuge for 30 sec.
8. Remove the spin column from the collection tube and discard the liquid. Replace spin column in the empty collection tube.
9. Centrifuge at 18,500 x g in a microcentrifuge for 30 sec.
10. Discard collection tube and liquid. Retain spin column.
11. Add 50 µl of DNA Elution Buffer to each spin column and place into a clean 1.5 ml tube.
12. Centrifuge at 18,500 x g in a microcentrifuge for 30 sec to elute DNA.
13. Remove and discard DNA spin column. Eluate is now purified DNA. (SAFE STOP) Samples can be stored at -20°C for up to 6 months.

B. DNA Purification Using Phenol/Chloroform Extraction and Ethanol Precipitation

NOTE: The following reagents are required for the phenol/chloroform extraction and ethanol precipitation and are not included in this kit: phenol/chloroform/isoamyl alcohol (25:24:1), chloroform/isoamyl alcohol (24:1), 3M Sodium Acetate (pH 5.2), 20mg/ml glycogen, 100% ethanol, 70% ethanol, and 1X TE buffer or nuclease-free water.

1. Add 300 µl of phenol/chloroform/isoamyl alcohol (25:24:1) to each input and enriched chromatin sample and mix thoroughly by vortexing for 30 sec.
2. Separate layers by centrifugation at 16,000 x g for 5 min in a microcentrifuge. Carefully transfer most of the top aqueous layer (avoiding the interphase) to a new tube.
3. Add 300 µl of chloroform/isoamyl alcohol (24:1) to the aqueous sample and mix thoroughly by vortexing for 30 sec.
4. Separate layers by centrifugation at 16,000 x g for 5 min in a microcentrifuge. Carefully transfer most of the top aqueous layer (avoiding the interphase) to a new tube.
5. Add 25 µl of 3M Sodium Acetate (pH 5.2), 1 µl 20mg/ml glycogen, and 600 µl of 100% ethanol to each aqueous sample and mix by vortexing for 30 sec.
6. Incubate samples at -80°C for 1 hr or -20°C overnight to precipitate DNA.
7. Pellet DNA by centrifugation at 16,000 x g for 5 min at 4°C in a microcentrifuge.
8. Carefully remove supernatant and wash pellet with 70% ethanol.
9. Pellet DNA by centrifugation at 16,000 x g for 5 min at 4°C in a microcentrifuge.
10. Decant supernatant and air dry pellet.
11. Resuspend pellet in 50 µl of 1X TE buffer or nuclease-free water. This is the purified DNA. (SAFE STOP) Samples can be stored at -20°C for up to 6 months.

VI. Quantification of DNA by qPCR

Recommendations:

• The Sample Normalization Primer Set included in the kit is specific for the S. cerevisiae ACT1 gene and can be used to quantify the signal from the Sample Normalization Spike-In yeast DNA for sample normalization (optional).
• The additional control primers included in the kit are specific for the human or mouse RPL30 gene (#7014 or #7015) and can be used for quantitative real-time PCR analysis of the Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb sample. If the user is performing CUT&RUN on another species, the user needs to design the appropriate control primers and determine the optimal PCR conditions for that species.
• PCR primer selection is critical. For CUT&RUN, PCR amplicon sizes should be approximately 60 to 80 bp in length. Primers should be designed with optimum melting temperature around 60°C and GC content around 50%.
• 2 µl of purified DNA is sufficient for qPCR-mediated quantification of target genes for histones, transcription factors, and cofactors.
• A Hot-Start Taq polymerase is recommended to minimize the risk of nonspecific PCR products.
• Use Filter-tip pipette tips to minimize risk of contamination.

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 tri-methyl-histone H3 Lys4 sample, the negative control rabbit IgG sample, a tube with no DNA to control for DNA contamination, and a serial dilution of the input DNA (undiluted, 1:5, 1:25, 1:125) to create a standard curve and determine the efficiency of amplification and quantify the amount of DNA in each immune-enriched sample.

   NOTE: If sample normalization is performed, only the CUT&RUN samples are to be analyzed using the Sample Normalization Primer Set. The input DNA does not contain the Normalization Spike-In DNA.

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. Set up 2-3 replicates for each PCR reaction. Add enough reagents to account for loss of volume (1-2 extra reactions). Add 18 µl of reaction mix to each PCR reaction tube or well.

4. Start the following PCR reaction program:

5. Analyze quantitative PCR results using the software provided with the real-time PCR machine. Alternatively, one can calculate the IP efficiency manually using the Percent Input Method and the equation shown below. With this method, signals obtained from each immunoprecipitation are expressed as a percent of the total input chromatin.
• Percent Input = 100% x 2^{(C[T] 100%Input Sample - C[T] IP Sample)}
• C[T] = C_T = Average threshold cycle of PCR reaction
6. For sample normalization, choose the sample that has the lowest C[T] value for the Sample Normalization Primer Set as the selected sample (e.g. Sample 1 in the example table below) and calculate the normalization factor of other samples using the below equation. Adjust the signals from the test primer sets using the respective normalization factors .

**Normalization Factor for qPCR = 2^{(C[T] Selected Sample - C[T] the Other Sample)}

FIGURE 8. Normalization of CUT&RUN signals using spike in DNA for qPCR analysis. CUT&RUN was performed with a decreasing number of HCT116 cells and either Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb #9751 (upper panels) or Phospho-Rpb1 CTD (Ser2) (E1Z3G) Rabbit mAb #13499 (lower panels). Enriched DNA was quantified by real-time PCR using SimpleChIP^® Human GAPDH Exon 1 Primers #5516, SimpleChIP^® Human β-Actin Promoter Primers #13653, SimpleChIP^® Human Β-Actin 3' UTR Primers #13669, and SimpleChIP^® Human MyoD1 Exon 1 Primers #4490. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin for 100,000 cells. Non-normalized enrichments are depicted in the left panels. The Sample Normalization Spike-In DNA was added into each reaction proportionally to the starting cell number. Based on the difference of qPCR signals from spike in DNA in each sample, CUT&RUN signals were normalized to the sample containing 100,000 cells. Normalized enrichments are depicted in the right panels.

VII. NG-Sequencing Library Construction

The immuno-enriched DNA samples prepared with this kit are directly compatible with 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^® platforms, we recommend using the SimpleChIP^® ChIP-seq DNA Library Prep Kit for Illumina^® #56795 with SimpleChIP^® ChIP-seq Multiplex Oligos for Illumina^® #29580 or #47538.

Additional Recommendations for DNA Library Preparation:

• During DNA End Preparation (Section I, Step 4 of the SimpleChIP^® ChIP-seq DNA Library Prep Kit for Illumina^® #56795), change the thermocycler program to incubate at 50^°C for 30 min instead of 65^°C for 30 min to avoid denaturation of small DNA fragments. Denatured DNA fragments are not compatible with adaptor ligation.
• When purifying adaptor-ligated DNA (Section III, Step 1 of the SimpleChIP^® ChIP-seq DNA Library Prep Kit for Illumina^® #56795) and library DNA (Section V, Step 1 of the SimpleChIP^® ChIP-seq DNA Library Prep Kit for Illumina^® #56795), add 1.1X volume instead of 0.9X volume of AMPure^® XP beads or SPRIselect^® beads to samples to increase the capture of smaller DNA fragments.
• During PCR Enrichment of adaptor-ligated DNA (Section IV, Step 3 of the SimpleChIP^® ChIP-seq DNA Library Prep Kit for Illumina^® #56795), reduce the Anneal and Extension time from 75 sec to 15 sec to exclude amplification of large library DNA fragments (>1,000 bp).
• The DNA yield from CUT&RUN assay may be lower than the 5 ng starting DNA we recommended for ChIP-seq library preparation. If the DNA yield is lower than 5 ng, we recommend increasing the number of PCR amplification cycles to 12-15 cycles in order to generate a library with DNA concentration of 10-30 ng/µl.
• Because of the very low background signal generated in CUT&RUN, a sequencing depth of 5 million reads per sample is usually sufficient for histone modifications and transcription factors. The duplication rate of reads significantly increases if the sequencing depth is greater than fifteen million per sample. The signal to noise ratio decreases if the sequencing depth is lower than two million per sample.
• When performing sample normalization, map CUT&RUN sequencing data for all samples to both the test reference genome (e.g. human) and the sample normalization S. cerevisiae yeast genome. Choose the sample that has the least number of unique yeast reads as the selected sample (e.g. Sample 1 in table below) and calculate the normalization factor of other samples using the equation below. Downsize the number of unique reads aligned to test reference genome for each sample using the respective normalization factors. Use the downsized dataset for further NGS analysis.

Normalization Factor for NGS = the number of unique yeast reads from Selected Sample / the number of unique yeast reads from the other sample

APPENDIX A: Determination of Cell Sensitivity to Digitonin

In the CUT&RUN protocol, the addition of digitonin to the buffers facilitates the permeabilization of cell membranes and entry of the primary antibody and pAG-MNase enzyme into the cells and nuclei. Therefore, having an adequate amount of digitonin in the buffers is critical to the success of antibody and enzyme binding and digestion of targeted genomic loci. Different cell lines show differing sensitivities to digitonin cell permeabilization. While the amount of digitonin recommended in this protocol should be sufficient for permeabilization of most cell lines, we recommend an initial test of your specific cell line. We have found that the addition of excess digitonin is not deleterious to the assay, so there is no need to perform a concentration curve. Rather, a quick test to determine if the recommended amount of digitonin works for your cell line is sufficient.

Before starting:

• Remove and warm Digitonin Solution at 90-100°C for 5 min. Make sure it is completely thawed. Immediately place the thawed Digitonin Solution on ice.

  NOTE: Digitonin Solution should be stored at -20°C. Please keep on ice during use and store at -20°C when finished for the day.

• For each cell line, prepare 100 µl of Digitonin Buffer (10 µl 10X Wash Buffer + 2.5 µl digitonin + 87.5 µl water). It is not necessary to add Spermidine or Protease Inhibitors for this test.

1. Collect 100,000 cells in a 1.5 ml tube.
2. Centrifuge for 3 min at 600 x g at room temperature and withdraw the liquid.
3. Resuspend cell pellet in 100 µl of Digitonin Buffer and incubate for 10 min at room temperature.
4. Mix 10 µl of cell suspension with 10 µl of 0.4% Trypan Blue Stain.
5. Use a hemocytometer or cell counter to count the number of stained cells and the total number of cells. Sufficient permeabilization results in > 90% of cells staining with Trypan blue.
6. If less than 90% of cells stain with Trypan blue, then increase the amount of Digitonin Solution added to the Digitonin Buffer and repeat steps 1-5 until > 90% cells are permeabilized and stained. Use this amount of Digitonin Solution in Sections I, II, and III.

APPENDIX B: Sonication Optimization for the Input Sample

Sonication of the input DNA sample is recommended because only fragmented genomic DNA (<10 kb) can be purified using DNA purification spin columns. Additionally, the fragmented genomic DNA (<1kb) may be used as the negative control in NG-seq analysis. Sonication should be optimized so that the input DNA is 100-600 bp in length.

We recommend using the input sample for NG-seq because it provides a convenient and unbiased representation of the cell genome. While the IgG sample can also be used as a negative control for NG-seq, it may show enrichment of specific regions of the genome due to non-specific binding. Unfragmented input DNA can be used for qPCR analysis. However, unfragmented DNA must be purified using phenol/chloroform extraction followed by ethanol precipitation.

Before starting:

! All buffer volumes should be increased proportionally based on the number of input samples being prepared.

• Remove and warm DNA Extraction Buffer at room temperature, making sure it's completely thawed and in solution.
• For each input sample, prepare 2.1 ml 1X Wash Buffer (210 µl 10X Wash Buffer + 1.89 ml water) and equilibrate it to room temperature to minimize stress on the cells. It is not necessary to add Spermidine or Protease Inhibitor Cocktail to this Wash Buffer.
• For each input sample, prepare 2 µl Proteinase K + 0.5 µl RNAse A to 197.5 µl DNA Extraction Buffer (200 µl per input sample).

1. In a 1.5 ml tube, collect 100,000 cells per each sonication condition.
2. Centrifuge for 3 min at 600 x g at room temperature and remove the liquid.
3. Resuspend cell pellet in 1 ml of 1X Wash Buffer by gently pipetting up and down.
4. Centrifuge for 3 min at 600 x g at room temperature and remove the liquid.
5. Wash the cell pellet again by repeating steps 3 and 4 one time.
6. For each 100,000 cells, add 100 µl of 1X Wash Buffer and resuspend the cell pellet by gently pipetting up and down.
7. Aliquot 100 µl cell suspension into a new tube for each sonication condition.

   NOTE: Samples will be incubated at 55°C in Step 9, so it is recommended to use a safe-lock 1.5 ml tube to reduce evaporation during the incubation.

8. Add 200 µl DNA Extraction Buffer (+ Proteinase K + RNAse A) to each sample and mix by pipetting up and down.
9. Place the tubes at 55°C for 1 hr with shaking.
10. Place the tubes on ice for 5 min to completely cool down the samples.
11. Determine optimal sonication conditions for your sonicator by setting up a time-course experiment with increasing numbers of 15 sec pulse sonication cycles. Be sure to incubate samples on ice for 30 sec between pulses.
12. Clarify lysates by centrifugation at 18,500 x g in a microcentrifuge for 10 min at 4°C. Transfer supernatant to a new 1.5 ml microcentrifuge tube.
13. Purify the DNA samples with DNA Purification Spin Columns or phenol/chloroform extraction followed by ethanol precipitation, following the directions in Section V.
14. Elute the DNA from the column or resuspend DNA pellet in 30 µl of 1X TE buffer or nuclease-free water.
15. Determine DNA fragment sizes by electrophoresis. Load > 15 µl sample on a 1% agarose gel with a 100 bp DNA marker. A dye-free loading buffer (30% glycerol) is recommended to better observe the DNA smear on gel.
16. Choose the sonication conditions that generate the optimal DNA fragment size of 100-600 bp and use for Preparation of the Input Sample in Section IV, Step 4. If optimal sonication conditions are not achieved, increase or decrease the power setting of the sonicator or number of sonication cycles and repeat the sonication time course experiment.

APPENDIX C: Troubleshooting Guide