RNF20 (D6E10) XP^® Rabbit mAb #11974

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:

Primer length:	24 nucleotides
Optimum Tm:	60°C
Optimum GC:	50%
Amplicon size:	150 to 200 bp (for standard PCR)
	80 to 160 bp (for real-time quantitative PCR)

Standard PCR Method

1. Label the appropriate number of 0.2 ml PCR tubes for the number of samples to be analyzed. These should include the 2% input sample, the positive control histone H3 sample, the negative control normal rabbit IgG sample, and a tube with no DNA to control for DNA contamination.
2. Add 2 µl of the appropriate DNA sample to each tube.
3. Prepare a master reaction mix as described below, making sure to add enough reagent for two extra tubes to account for loss of volume. Add 18 µl of master mix to each reaction tube.

Reagent	Volume for 1 PCR Reaction (18 µl)
Nuclease-free H2O	12.5 µl
10X PCR Buffer	2.0 µl
4 mM dNTP Mix	1.0 µl
5 µM RPL30 Primers	2.0 µl
Taq DNA Polymerase	0.5 µl

4. Start the following PCR reaction program:

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. 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.

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.

   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 DNA Library Prep Kit for Illumina^® (ChIP-seq, CUT&RUN) #56795 and its associated index primers Multiplex Oligos for Illumina^® (Single Index Primers) (ChIP-seq, CUT&RUN) #29580 or Multiplex Oligos for Illumina^® (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 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.

Tissue/Cell	Total Chromatin Yield	Expected DNA Concentration
Spleen	20-30 µg per 25 mg tissue	200-300 µg/ml
Liver	10-15 µg per 25 mg tissue	100-150 µg/ml
Kidney	8-10 µg per 25 mg tissue	80-100 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
HeLa	10-15 µg per 4 x 106 cells	100-150 µg/ml

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

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

CUT&RUN Protocol

I. Cell and Tissue Preparation

For most cell types, live cells can be used in the CUT&RUN assay to generate robust enrichment of histones, transcription factors, and cofactors. For certain cell types that are fragile or sensitive to Conconavalin A, a light cell fixation helps to preserve the cells and keep them intact. In addition, fixation may help to boost enrichment of low abundance and/or weak binding transcription factors and cofactors if robust signal is not observed using fresh cells. Please note that over-fixation of cells will inhibit the CUT&RUN assay.

Our CUT&RUN assay works with a wide range of cell or tissue inputs. As defined in the protocol, one CUT&RUN reaction can contain between 5,000 to 250,000 cells or 1 to 5 mg of tissue. Buffer volumes used throughout the protocol do not need to be adjusted based on the amount of cells or tissue per reaction, as long as it is within this range. When indicated, buffer volumes do need to be increased proportionally based on the number of reactions being performed. If possible, we recommend using 100,000 cells or 1 mg of tissue per reaction. If cells are limiting, we recommend using at least 5,000 to 10,000 cells per reaction for histone modifications and 10,000 to 20,000 cells per reaction for transcription factors or cofactors.

NOTE: The amount of digitonin recommended for cell permeabilization is in excess and should be sufficient for permeabilization of most cell lines and tissue types. However, not all cell lines and tissues exhibit the same sensitivity to digitonin. If your specific cell line or tissue does not work with the recommended digitonin concentration, you can optimize conditions by following the protocol provided in Appendix A. Digitonin treatment should result in permeabilization of >90% of the cell population.

A. Live Cell Preparation

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 #7012 and 100X Spermidine #27287. Make sure both are completely thawed.
• 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 #31415 + 25 µl 100X Spermidine #27287 + 12.5 µl 200X Protease Inhibitor Cocktail #7012 + 2212.5 µl Nuclease-free Water #12931. Equilibrate it to room temperature to minimize stress on the cells.

  NOTE: Steps for live cell (no fixation) preparation should be performed in succession at room temperature to minimize stress on the cells. To minimize DNA fragmentation, avoid vigorous vortexing and cavitation of samples during resuspension. When preparing live cells for CUT&RUN, we recommend preparing the Concanavalin A Beads (Section II, Steps 1 to 5) prior to preparing the cells as to minimize the amount of time the cells sit around during bead preparation. Activated beads can be stored on ice until ready to use.

1. Harvest fresh cell cultures at room temperature to minimize stress on the cells. Collect 5,000 to 100,000 cells for each reaction and an additional 5,000 to 100,000 cells for the input sample. Be sure to include reactions for the positive control Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb #9751 and the negative control Rabbit (DA1E) mAb IgG XP^® Isotype Control (CUT&RUN) #66362.

   NOTE: For adherent cells, the cells first need to be detached from the dish using Trypsin and neutralized with at least 3 volumes of tissue culture medium. We do not recommend scraping the cells from the dish because this can stress and even lyse the cells. Cells should be counted using a hemocytometer or other cell counter to ensure the proper number of cells are being used for the experiment.

2. Centrifuge cell suspension for 3 min at 600 x g at room temperature and remove the liquid.

   NOTE: The challenge of working with low cell numbers (<100,000 total cells) is that the centrifuged cell pellet is not always visible by eye, making it easy to lose cells during the wash steps. Therefore, when working with low cell numbers, we recommend skipping the wash steps 3 to 5 below. The binding of the Concanavalin A beads to cells is tolerant to having 40% cell medium in the binding reaction. Therefore, after the initial centrifugation of the cell suspension in Step 2, one can remove most of the supernatant, leaving behind ≤40 µl cell medium per reaction. Then in Step 6 add enough 1X Wash Buffer (+ spermidine + PIC) to the cell suspension to achieve a total volume of 100 µl per reaction.

3. Resuspend cell pellet in 1 ml of 1X Wash Buffer (+ spermidine + PIC) at room temperature 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 a second time by repeating steps 3 and 4 one time.
6. For each reaction or input sample, add 100 µl of 1X Wash Buffer (+ spermidine + PIC) and resuspend the cell pellet by gently pipetting up and down.
7. Transfer 100 µl of cells to a new tube and store at 4°C until Section V. This is the 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.

8. Immediately proceed to Section II.

B. Fixed Cell Preparation

NOTE: The following reagents are required for fixed cell preparation and are not included in this kit: 37% formaldehyde or 16% Formaldehyde Methanol-Free #12606, Glycine Solution (10X) #7005, and 10% SDS Solution #20533.

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 #7012 and 100X Spermidine #27287. Make sure both are completely thawed.
• 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 #31415 + 25 µl 100X Spermidine #27287 + 12.5 µl 200X Protease Inhibitor Cocktail #7012 + 2212.5 µl Nuclease-free Water #12931. Equilibrate it to room temperature to minimize stress on the cells.
• Prepare 2.7 µl of 37% formaldehyde or 6.25 µl of 16% Formaldehyde Methanol-Free #12606 per 1 ml of cell suspension to be processed and keep at room temperature. Use fresh formaldehyde that is not past the manufacturer’s expiration date.

1. Collect 5,000 to 100,000 cells for each antibody/MNase reaction and an additional 5,000 to 100,000 cells for the input sample. Be sure to include reactions for the positive control Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb #9751 and the negative control Rabbit (DA1E) mAb IgG XP^® Isotype Control (CUT&RUN) #66362.

   NOTE: With adherent cell lines, cells first need to be detached from the dish using Trypsin and neutralized with at least 3 volumes of medium. We don’t recommend scraping the cells from the dish because this can stress and even lyse the cells. Cells should be counted using a hemocytometer or other cell counter to ensure the proper number of cells are being used for the experiment.

2. Add 2.7 µl 37% formaldehyde or 6.25 µl 16% Formaldehyde Methanol-Free #12606 per 1 ml of cell suspension to achieve a final concentration of 0.1% formaldehyde. Swirl tube to mix and incubate at room temperature for 2 min.
3. Stop cross-linking by adding 100 µl of Glycine Solution (10X) #7005 per 1 ml of fixed cell suspension. Swirl the tube to mix and incubate at room temperature for 5 min.
4. Centrifuge cell suspension for 3 min at 3,000 x g at 4°C and remove the liquid. Immediately proceed to Step 5. (SAFE STOP) Alternatively, fixed cell pellets may be stored at -80°C before using for up to 6 months.

   NOTE: The challenge of working with low cell numbers (<100,000 total cells) is that the centrifuged cell pellet is not always visible by eye, making it easy to lose cells during the wash steps. In this case we do NOT recommend freezing down cell pellets. In addition, when working with these low cell numbers, we recommend skipping the wash steps 5 to 7 below. The binding of the Concanavalin A beads to cells is tolerant to having 40% cell medium in the binding reaction. Therefore, after the initial centrifugation of the cell suspension in Step 4, one can remove most of the supernatant, leaving behind ≤40 µl cell medium per reaction. Then in Step 8 add enough 1X Wash Buffer (+ spermidine + PIC) to the cell suspension to achieve a total volume of 100 µl per reaction.

5. Resuspend cell pellet in 1 ml of 1X Wash Buffer (+ spermidine + PIC) by gently pipetting up and down.
6. Centrifuge for 3 min at 3,000 x g at 4°C and remove the liquid.
7. Wash the cell pellet a second time by repeating steps 5 and 6 one time.
8. For each reaction or input sample, add 100 µl of 1X Wash Buffer (+ spermidine + PIC) and resuspend the cell pellet by gently pipetting up and down.
9. Transfer 100 µl of cells to a new tube and store at 4°C until Section V. This is the 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.

10. Immediately proceed to Section II.

C. Tissue Sample Preparation

For most tissue types, 1 mg of lightly fixed tissue (0.1% formaldehyde for 2 min) can generate robust enrichment of histones, transcription factors and cofactors. Formaldehyde fixation is not required for enrichment of histone modifications. However, many transcription factors and cofactors do require light fixation of the tissue for optimal results. Some low abundance and/or weak binding transcription factors and cofactors may require a medium fixation (0.1% formaldehyde for 10 min) for optimal results. In addition, medium fixation may improve results when using difficult tissue types, like fibrous tissues. Please note that over-fixation will inhibit the CUT&RUN assay. Fixed tissue samples can be frozen at -80°C for up to 6 months before using.

NOTE: When preparing fresh tissue (no fixation) for CUT&RUN, we recommend preparing the Concanavalin A Beads (Section II, Steps 1 to 5) prior to preparing the tissue as to minimize the amount of time the cells sit around during bead preparation. Activated beads can be stored on ice until ready to use.

NOTE: The following reagents are required for fixed tissue preparation and are not included in this kit: 37% formaldehyde or 16% Formaldehyde Methanol-Free #12606, Phosphate Buffered Saline (PBS) #9872, Glycine Solution (10X) #7005, and 10% SDS Solution #20533.

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 #7012 and 100X Spermidine #27287. Make sure both are completely thawed.
• Prepare 1X Wash Buffer (3 ml for each tissue type and additional 100 µl for each reaction or input sample). For example, to prepare 3.5 ml of 1X Wash Buffer, add 350 µl 10X Wash Buffer #31415 + 35 µl 100X Spermidine #27287 + 17.5 µl 200X Protease Inhibitor Cocktail #7012 + 3097.5 µl Nuclease-free Water #12931. Equilibrate it to room temperature to minimize stress on the cells.
• Prepare the following buffers if tissue fixation is needed:
  • Prepare 1 ml fixation buffer for each tissue type by adding 2.7 µl of 37% formaldehyde or 6.25 µl of 16% Formaldehyde Methanol-Free #12606 and 5 µl 200X Protease Inhibitor Cocktail (PIC) #7012 into 1 ml of Phosphate Buffered Saline (PBS) #9872. Use fresh formaldehyde that is not past the manufacturer’s expiration date.
  • Prepare 1 ml of PBS #9872 + 5 µl Protease Inhibitor Cocktail (PIC) #7012 for each tissue type and place on ice.
  • Prepare 100 µl of Glycine Solution (10X) #7005 per 1 ml of fixation buffer.

1. Weigh 1 mg fresh tissues for each antibody/MNase reaction and an additional 1 mg of tissue for the input sample. Be sure to include reactions for the positive control Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb #9751 and the negative control Rabbit (DA1E) mAb IgG XP^® Isotype Control (CUT&RUN) #66362.

   NOTE: For some transcription factors or cofactors, or for difficult tissue types like fibrous tissues, up to 5 mg tissue per reaction can be used without scaling up reagents.

2. Place tissue sample in a 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.

   NOTE: We recommend light fixation of tissues because this condition works optimally for most tissue types and protein targets. However, if fresh tissues are desired, skip Steps 3 to 8 and immediately proceed to Step 9.

3. Immediately transfer minced tissue to 1 ml of fixation solution and swirl tube to mix.

   NOTE: This volume of fixation solution is sufficient for up to 50 mg of tissue. If processing >50 mg, scale up fixation solution and 1X PBS + PIC solution in Step 7 accordingly.

4. Incubate at room temperature for 2 min.

   NOTE: For difficult tissue types (like fibrous tissues) or low abundance and/or weak binding transcription factors or cofactors, extending the formaldehyde fixation to 10 min may improve results.

5. Stop cross-linking by adding 100 µl of Glycine Solution (10X) #7005 per 1 ml of fixation buffer. Swirl the tube to mix and incubate at room temperature for 5 min.
6. Centrifuge tissue for 5 min at 2,000 x g at 4°C and remove the liquid.
7. Resuspend tissue with 1 ml of 1X PBS + PIC.
8. Centrifuge for 5 min at 2,000 x g at 4°C and remove the liquid and proceed to step 9. (SAFE STOP) Alternatively, fixed tissue pellets may be stored at -80°C before disaggregation for up to 6 months.
9. Resuspend tissue in 1 ml of 1X Wash Buffer (+ spermidine + PIC) and transfer the sample to a Dounce homogenizer.
10. Disaggregate tissue pieces into single-cell suspension with 20-25 strokes until no tissue chunks are observed.
11. Transfer cell suspension to a 1.5 ml tube and centrifuge at 3,000 x g for 3 min at room temperature, remove supernatant from cells.
12. Resuspend cell pellet in 1 ml of 1X Wash Buffer (+ spermidine + PIC).
13. Centrifuge cell suspension for 3 min at 3,000 x g at room temperature and remove the liquid.
14. Wash the cell pellet a second time by repeating steps 12 and 13 one time.
15. For each reaction, add 100 µl of 1X Wash Buffer (+ spermidine + PIC) and resuspend the cell pellet by gently pipetting up and down.
16. Transfer 100 µl of cells to a new tube and store at 4°C until Section V. This is the 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.

17. Immediately proceed to Section II.

II. Binding of Concanavalin A Beads and Primary Antibody

Before Starting:

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

• Place Concanavalin A Bead Activation Buffer on ice.
• For each reaction, prepare 1 µl 100X Spermidine #27287 + 0.5 µl 200X Protease Inhibitor Cocktail #7012 + 2.5 µl Digitonin Solution #16359 + 96 µl Antibody Binding Buffer #15338 and place on ice (100 µl per reaction).

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 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 pipet. 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 one time.
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.

   NOTE: If Concanavalin A Beads are prepared prior to cell or tissue preparation, as recommended for live cells and fresh tissue, the activated beads can be stored on ice until use.

6. Make sure Concanavalin A Beads are mixed well into solution. Add 10 µl of activated bead suspension per reaction to the washed cell suspension prepared in Section I-A Step 8, Section I-B Step 10, or Section I-C Step 17.
7. Mix the samples well by pipetting up and down. Incubate 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.

8. Briefly centrifuge the sample at 100 x g for 2 sec 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.
9. Remove tube from the stand. Add 100 µl of Antibody Binding Buffer (+ spermidine + PIC + digitonin) per reaction and place on ice.
10. Aliquot 100 µl of the cell:bead suspension into separate 1.5 ml tubes for each reaction and place on ice.
11. 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 #9751, 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, when possible.

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

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

III. 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 #16359 to 90-100°C for 5 min and make sure it is completely thawed and in solution. Immediately place the thawed Digitonin Solution #16359 on ice.

  NOTE: Digitonin Solution #16359 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 #31415 + 10.5 µl 100X Spermidine #27287 + 5.25 µl 200X Protease Inhibitor Cocktail #7012 + 26.25 µl Digitonin Solution #16359 + 903 µl Nuclease-free Water #12931).
• 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 II, Step 12 at 100 x g for 2 sec 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, make sure to collect all beads that are stuck to the tube wall.
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. Incubate tubes at 4°C for 1 hr.

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

7. Immediately proceed to Section IV.

IV. 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 #16359 to 90-100°C for 5 min and make sure it is completely thawed and in solution. Immediately place the thawed Digitonin Solution #16359 on ice.

  NOTE: Digitonin Solution #16359 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 #31415 + 21.5 µl 100X Spermidine #27287 + 10.75 µl 200X Protease Inhibitor Cocktail #7012 + 53.75 µl Digitonin Solution #16359 + 1.849 ml Nuclease-free Water #12931).
• Place Calcium Chloride on ice.
• If starting with fixed materials in Section I, make sure the 10% SDS Solution #20533 is completely in solution. Warming it up to 37°C will help to dissolve the SDS precipitates.
• For each reaction, prepare 150 µl of 1X Stop Buffer (37.5 µl 4X Stop Buffer #48105 + 3.75 µl Digitonin Solution #16359 + 0.75 µl RNAse A #7013 + 108 µl Nuclease-free Water #12931).

  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 VII). 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 100-fold into Nuclease-free Water #12931 and then adding 5 µl (50 pg) of Spike-In DNA to each reaction. When using 100,000 cells or 1 mg of tissue per reaction this ensures that the normalization reads are around 0.5% of the total sequencing reads. If more or less than 100,000 cells or 1 mg of tissue are used per reaction, proportionally scale the volume of Sample Normalization Spike-In DNA up or down to adjust normalization reads to around 0.5% of total reads.

1. Briefly centrifuge samples from Section III, Step 6 at 100 x g for 2 sec 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 pAG-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.
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 supernatants to new 2 ml microcentrifuge tubes. These are your enriched chromatin samples.

    NOTE: If live cells or fresh tissues (not fixed) are used for the CUT&RUN assay, skip Steps 14-15 and immediately proceed to Step 16.

    NOTE: Fixed samples will be incubated at 65°C later in the protocol, so it is recommended to use a safe-lock 2 ml tube to reduce evaporation during the incubation.

14. To reverse the crosslinks in fixed cell or tissue samples, allow samples to warm to room temperature and add 3 µl of 10% SDS Solution #20533 (0.1% final concentration) and 2 µl of proteinase K (20 mg/ml) #10012 to each sample.

    NOTE: SDS may precipitate out of solution if samples are not pre-warmed to room temperature.

15. Vortex each sample and incubate at 65°C for at least 2 hr. This incubation can be extended overnight. After incubation, quickly spin samples at 10,000 x g for 1 sec to collect evaporation from the cap of tubes.
16. Equilibrate samples to room temperature and proceed to Section VI. (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 VI).

V. Preparation of the Input Sample

Fragmentation of input DNA is required for compatibility with downstream NG-Sequencing but is not necessary for downstream qPCR analysis. If a sonicator is not available, we recommend using the unfragmented input DNA for qPCR analysis; however, the input DNA should be purified using phenol/chloroform extraction and ethanol precipitation because the size of unfragmented input DNA is too large to be purified using DNA spin columns. If a sonicator is not available and downstream NG-Sequencing analysis is desired, one can use the CUT&RUN normal IgG antibody sample as the negative control, although this is not ideal because the normal IgG-enriched sample may show non-specific DNA enrichment. Alternatively, an input DNA fragmentation protocol using MNase is available at https://cst-science.com/CUT-RUN-input-digestion.

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

Before Starting:

• Remove and warm DNA Extraction Buffer #42015. Make sure it is completely thawed.
• For each input sample, prepare 2 µl Proteinase K #10012 + 0.5 µl RNAse A #7013 + 197.5 µl DNA Extraction Buffer #42015 (200 µl total per input sample).

1. Add 200 µl of DNA Extraction Buffer (+ Proteinase K + RNAse A) to the 100 µl input sample from Section I-A Step7, Section I-B Step 9, or Section I-C Step 16. 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 2 ml microcentrifuge tube.
6. Immediately proceed to Section VI 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 VI).

VI. DNA Purification

DNA can be purified from input and enriched chromatin samples using DNA spin columns, as described in Section VI - A, or phenol/chloroform extraction followed by ethanol precipitation as described in Section VI - 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 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 before use. This step only has to be performed once prior to the first set of DNA purifications.
• Remove one DNA Purification collection tube 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 mix by pipetting up and down.

   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 #12931.

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 #12931. This is the purified DNA. (SAFE STOP) Samples can be stored at -20°C for up to 6 months.

VII. 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 #9751 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.

Reagent	Volume for 1 PCR Reaction (18 µl)
Nuclease-free H2O #12931	6 µl
5 µM Primers	2 µl
SimpleChIP® Universal qPCR Master Mix #88989	10 µl

4. Start the following PCR reaction program:

a.	Initial Denaturation	95°C for 3 min
b.	Denature	95°C for 15 sec
c.	Anneal and Extension	60°C for 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.
• 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.

An Example of Sample Normalization for qPCR Assay (see Figure 8)

	C[T] value of Sample Normalization Primer Set	**Normalization Factor for qPCR	Signal Before Normalization (% Input Calc'd from Step 5)	Signal After Normalization
Sample 1	23.31	2(23.31-23.31)=1.00	24.4%	24.4%/1.00=24.4%
Sample 2	24.24	2(23.31-24.24)=0.52	12.0%	12.0%/0.52=23.1%
Sample 3	25.08	2(23.31-25.08)=0.29	6.28%	6.28%/0.29=21.7%
Sample 4	26.30	2(23.31-26.30)=0.13	2.72%	2.72%/0.13=20.9%

**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.

VIII. 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 DNA Library Prep Kit for Illumina^® (ChIP-seq, CUT&RUN) #56795 with Multiplex Oligos for Illumina^® (ChIP-seq, CUT&RUN) #29580 or #47538, following the Protocol for CUT&RUN DNA.

• 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.
• For less than 20,000 starting number of cells, it is common to obtain lower mapping rates or higher duplication rates in the NGS reads. If this happens, we recommend increasing the sequencing depth to obtain a sufficient amount of unique mapped reads for downstream data analysis.
• 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.

An Example of Sample Normalization for NGS Assay

	The Number of Unique Reads Aligned to Yeast	Normalization Factor for NGS	The Number of Unique Reads Aligned to Test Reference Genome Before Normalization	The Number of Unique Reads Aligned to Test Reference Genome After Normalization
Sample 1	219,275	219,275/219,275 = 1.00	5,077,747	5,077,747 X 1.00 = 5,077,747
Sample 2	411,915	219,275/411,915 = 0.53	9,896,671	9,896,671 X 0.53 = 5,268,306
Sample 3	816,235	219,275/816,235 = 0.27	17,842,773	17,842,773 X 0.27 = 4,793,320
Sample 4	1,120,826	219,275/1,120,826 = 0.20	23,836,679	23,836,679 X 0.20 = 4,663,339

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 or tissues, you can test your specific cell line or tissue using this protocol. 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 #16359 to 90-100°C for 5 min. Make sure it is completely thawed. Immediately place the thawed Digitonin Solution #16359 on ice.

  NOTE: Digitonin Solution #16359 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 or tissue sample, prepare 100 µl of Wash Buffer (10 µl 10X Wash Buffer #31415 + 90 µl Nuclease-free Water #12931). It is not necessary to add spermidine or Protease Inhibitors for this test.

1. In a 1.5 ml tube, collect 10,000 - 100,000 cells. For tissue, collect disaggregated cells from 1 mg of tissue (Section I-C Step 1-13). If you are using fixed cells or tissue in your CUT&RUN experiment, be sure to fix the cells or tissue the same way for this test.
2. Centrifuge for 3 min at 600 x g at room temperature and withdraw the liquid.

   NOTE: If the cell pellet is not visible by eye, we recommend removing as much cell medium as possible without disturbing the cell pellet after the initial centrifugation of the cell suspension in Step 2 and leave behind some cell medium per reaction. Then in Step 3 add enough 1X Wash Buffer to the cell suspension to achieve a total volume of 100 µl.

3. Resuspend cell pellet in 100 µl of Wash Buffer.
4. Add 2.5 µl Digitonin Solution #16359 to each reaction and incubate for 10 min at room temperature.
5. Mix 10 µl of cell suspension with 10 µl of 0.4% Trypan Blue Stain.
6. 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.
7. If less than 90% of cells stain with Trypan blue, then increase the amount of Digitonin Solution #16359 added to the Digitonin Buffer and repeat steps 1-5 until > 90% cells are permeabilized and stained. Use this amount of Digitonin Solution #16359 in Sections I - IV.

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 #42015 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 #31415 + 1.89 ml Nuclease-free Water #12931) and equilibrate it to room temperature to minimize stress on the cells. It is not necessary to add spermidine or Protease Inhibitor Cocktail #7012 to this Wash Buffer.
• For each input sample, prepare 2 µl Proteinase K #10012 + 0.5 µl RNAse A #7013 to 197.5 µl DNA Extraction Buffer #42015 (200 µl per input sample).

1. In a 1.5 ml tube, collect the same number of cells you use for the input in your CUT&RUN experiment (5,000 to 100,000 cells) for each sonication condition being tested. For tissue, collect disaggregated cells from the same amount of tissue you use for the input in your CUT&RUN experiment (Section I-C Step 1-13) for each sonication condition being tested. If you are using fixed cells or tissue in your experiment, be sure to fix the cells or tissue the same way for this test.
2. Centrifuge for 3 min at 600 x g at room temperature and remove the liquid.

   NOTE: If the centrifuged cell pellet is not visible by eye when working with low cell numbers (<100,000 cells), we recommend skipping the wash steps 3-5 below. Remove as much cell medium as possible without disturbing the cell pellet after the initial centrifugation of the cell suspension in Step 2 and leave behind some cell medium per reaction. Then in Step 6 add enough 1X Wash Buffer to the cell suspension to achieve a volume of 100 µl per sonication condition being tested.

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. Add 100 µl of 1X Wash Buffer per sonication condition being tested and resuspend the cell pellet by gently pipetting up and down.
7. Aliquot 100 µl of the 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 2 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 VI.
14. Elute the DNA from the column or resuspend DNA pellet in 30 µl of 1X TE buffer or Nuclease-free Water #12931.
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 V, 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

For a detailed troubleshooting guide, please go to https://cst-science.com/troubleshooting-CUT-RUN