CUT&RUN Assay Kit #86652

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.

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.

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.

An Example of Sample Normalization for NGS Assay (see Figure 9)

	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, 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

Problem	Possible Causes	Recommendation
1. Concanavalin A beads clump during the experiment.	Bead clumping is normal and is not usually deleterious to the assay.	Resuspend clumped beads by gently pipetting up and down.
	Room temperature incubation of beads and cells is too long.	Activate Concanavalin A beads at 4°C and incubate with cells no longer than 5 min (Section I, Step 14).
	Cells are lysing during preparation.	Be sure to prepare cells at room temperature and as quickly as possible to minimize cell stress (Section I, Steps 7-16).
	Digitonin concentration may be too high.	Some cells may be more sensitive to digitonin and lyse at higher concentrations. Reduce the amount of digitonin in the assay, but be sure to confirm the amount used is sufficient for cell permeabilization (see APPENDIX A).
2. No DNA is detected in the purified DNA samples using a picogreen-based DNA quantification assay.	This is typical when starting with low cell numbers (<10,000 cells), but DNA should be detectable when starting with the recommended 100,000 cells.	Be sure to use a picogreen-based DNA quantification assay. Purified DNA is not typically detectable using a NanoDrop, Bioanalyzer® or Tapestation®.
	Cell count is off, cells are lost or lysing during preparation.	Starting cell culture should be 60-90% confluent and look healthy (> 90% live cells).
		Be sure to prepare cells at room temperature and as quickly as possible to minimize cell stress.
		Wash all cells in one vial to minimize cell loss (Section I, Steps 7-16).
	Digitonin is not effectively permeabilizing the cells.	Be sure to store Digitonin Solution at -20°C when not in use, as it is unstable when stored above -20°C.
		Be sure to test and confirm that the amount of digitonin used is sufficient to permeabilize your specific cell line (see APPENDIX A).
	pAG-MNase enzyme is not working properly in the assay.	The pAG-MNase is highly stable and should maintain activity for a long time when stored properly.
		The pAG-MNase requires Ca2+ divalent cations for activity. Be sure to add calcium chloride for activation of the enzyme (Section III, Step 8).
		Be sure to digest for 30 min to allow the enzyme to sufficiently digest the chromatin (Section III, Step 9).
	Not enough antibody is added to the reaction or antibody does not work in the CUT&RUN assay.	Not all antibodies work in CUT&RUN. If possible, use a CUT&RUN validated antibody. Alternatively, some ChIP- and IF-validated antibodies also work for CUT&RUN.
		Be sure to include the positive control Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb to show your assay is working.
3. No signal in qPCR or NG-seq analysis.	See possible causes for problem #2.	See recommendations problem #2.
	Not enough DNA added to the qPCR reaction.	Add more DNA to the PCR reaction or increase the number of amplification cycles.
	Not enough DNA added to the NG-seq DNA library preparation.	Be sure to quantify the purified DNA using a picogreen-based DNA quantification assay and use the recommended amount of starting DNA and PCR-amplification cycles (see Section VII).
	PCR-amplified region may span a nucleosome-free region.	DNA fragments generated in the CUT&RUN assay are typically smaller than DNA fragments generated in the ChIP assay. Therefore, it is critical to design primers to generate amplicons no longer than 60 to 80 bp.
4. High background signal in qPCR or NG-seq analysis.	Genomic DNA has become highly fragmented due to harsh treatment of samples.	Always use the Rabbit (DA1E) mAb IgG XP® Isotype Control (CUT&RUN) #66362 negative control antibody to determine background signal in the CUT&RUN assay.
		To minimize DNA fragmentation, avoid vigorous vortexing and introduction of bubbles during resuspension of cells.
	Genomic DNA has become highly fragmented due to cell stress and lysis.	Be sure to prepare cells at room temperature and as quickly as possible to minimize cell stress. Wash all cells in one vial to minimize cell loss (Section I, Steps 7-16).
	Digestion is not performed at 0°C and chromatin is over-digested.	Digestion should be performed in an ice-water bath. Digestion at higher temperatures can significantly increase background signal.
		Make sure to pre-cool cell samples and calcium chloride in an ice-water bath for 5 min prior to adding the calcium chloride and initiating the digest. Quickly mix the samples and place back in the ice-water bath.
	Large non-specific genomic DNA can also diffuse into the supernatant and contaminate the smaller fragments released by targeted digestion.	Do not incubate samples at 37°C for > 10 min and do not shake samples during incubation (Section III, Step 11). Ten minutes is sufficient for diffusion of digested fragments into the supernatant.
		Large genomic DNA fragments can be removed by size-selection using AMPure® XP Beads or SPRIselect® Reagent Kit prior to qPCR analysis.
		For NG-seq analysis, shorten the PCR amplification time (10-15 sec) during library construction to exclude amplification of large DNA fragments.
	Too much antibody is used in the assay, resulting in non-specific binding and digestion.	If possible, be sure to use a CUT&RUN validated antibody at the recommended dilution. If not, ChIP-validated and IF-validated antibodies often work at their ChIP- and IF-recommended dilutions. You may need to titrate your antibody in the assay.