CUT&Tag Troubleshooting Guide

Select the CUT&Tag Problem you are EncounteringConcanavalin A beads clump during the experimentLow yield, low sequencing depth, or no signal in NG-seq analysisNG-seq exhibits signal in unpredicted genome regions

A. Fixed Cell Preparation

We strongly recommend using live cells whenever possible. For certain cell types that are fragile or sensitive to concanavalin A, please refer to the protocol below to lightly fix cells prior to the CUT&Tag experiment. Please note that cell fixation does not significantly increase CUT&Tag signals. In fact, over-fixation may lead to weaker CUT&Tag signals. Refer to the description in the CUT&Tag Protocol, Section II to determine the appropriate cell number in each reaction.

NOTE: The following reagents are required for fixed cell preparation and are not included in the CUT&Tag Assay Kit #77552: 37% formaldehyde or 16% Formaldehyde Methanol-Free #12606 and Glycine Solution (10X) #7005.

Before starting:

! All buffer volumes should be scaled proportionally to the number of CUT&Tag reactions being performed.

• 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.
• Thoroughly thaw 200X Protease Inhibitor Cocktail #7012 and 100X Spermidine #27287 before use and store them at -20°C when finished for the day. Please note that the Protease Inhibitor Cocktail #7012 will refreeze when placed on ice due to containing DMSO.
• Prepare Complete Wash Buffer (2 ml for each cell preparation and an additional 100 µl for each CUT&Tag reaction) and keep it at room temperature. For example, if using both untreated and drug-treated cells (2 cell preparations) and testing with 4 antibodies (positive control H3K4me3 #9751, negative control IgG #2729 or #68860, and two experimental antibodies; 8 reactions), a total of 4.8 ml of Complete Wash Buffer will be needed.

Complete Wash Buffer	Volume (per cell preparation)	Volume (per reaction)	Total Volume
10X Wash buffer (CUT&RUN, CUT&Tag) #31415	200 µl	10 µl	Add both columns together for total volume needed for each reagent.
100X Spermidine #27287	20 µl	1 µl
Protease Inhibitor Cocktail (200X) #7012	10 µl	0.5 µl
Nuclease free water #12931	1770 µl	88.5 µl

1. Harvest 100,000 live cells for each reaction.

NOTE: For adherent cells, detach them from the dish using Trypsin and neutralize with at least 3 volumes of tissue culture medium. We do not recommend scraping the cells from the dish to prevent cell lysis. Cells should be counted accurately 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 of 37% formaldehyde or 6.25 µl of 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 supernatant. 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: If working with fewer than 100,000 total cells and the centrifuged cell pellet is not visible by eye, we do NOT recommend freezing down cell pellets. Instead, we recommend continuing on with the protocol and skipping the wash steps 5 to 7 below. After the initial centrifugation of the cell suspension in Step 4, remove most of the supernatant, leaving behind ≤40 µl cell medium per reaction. Then in Step 8 add enough Complete Wash Buffer to the cell suspension to achieve a total volume of 100 µl per reaction.

5. Resuspend cell pellet in 1 ml of Complete Wash Buffer by gently pipetting up and down.
6. Centrifuge for 3 min at 3,000 x g at 4°C and remove the supernatant
7. Wash the cell pellet a second time by repeating steps 5 and 6 one time.
8. For each reaction, add 100 µl of Complete Wash Buffer and resuspend the cell pellet by gently pipetting up and down.
9. Immediately proceed to CUT&Tag Protocol, Section III.

B. Fixed Tissue Sample Preparation

For most tissue types, 1 mg of fresh tissue is sufficient to generate robust enrichment of histones. If fresh tissue is not accessible, lightly fixed tissue (0.1% formaldehyde for 2 min) can be used. Fixed tissue samples can be frozen at -80°C up to 6 months before using. Over-fixation may lead to weaker CUT&Tag signals. The CUT&Tag assay does not work well for enrichment of transcription factors and cofactors from tissues. For analysis of transcription factors and cofactors, we recommend using the CUT&RUN Assay Kit #86652.

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, and Glycine Solution (10X) #7005.

Before starting:

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

• 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.
• Prepare 100 µl of Glycine Solution (10X) #7005 per 1 ml of fixation buffer.
• Thoroughly thaw 200X Protease Inhibitor Cocktail #7012 and 100X Spermidine #27287 before use and store them at -20°C when finished for the day. Please note that the Protease Inhibitor Cocktail #7012 will refreeze when placed on ice due to containing DMSO.
• Prepare Complete Wash Buffer (3 ml for each tissue type and additional 100 µl for each reaction) and keep it at room temperature to minimize stress on the cells.

Complete Wash Buffer	Volume (per cell preparation)	Volume (per reaction)	Total Volume
10X Wash buffer (CUT&RUN, CUT&Tag) #31415	300 µl	10 µl	Add both columns together for total volume needed for each reagent.
100X Spermidine #27287	30 µl	1 µl
Protease Inhibitor Cocktail (200X) #7012	15 µl	0.5 µl
Nuclease free water #12931	2655 µl	88.5 µl

• Prepare 1 ml Fixation Buffer for each tissue type. Use fresh formaldehyde that is not past the manufacturer's expiration date.

Fixation Buffer	Volume (per tissue type)
Formaldehyde	2.7 µl of 37% or 6.25 µl of 16%
Protease Inhibitor Cocktail (200X) #7012	5 µl
Phosphate Buffered Saline (PBS) #9872	992.3 µl

• Prepare 1 ml of Fixation Wash Buffer for each tissue type and place on ice.

Fixation Wash Buffer	Volume (per tissue type)
Protease Inhibitor Cocktail (200X) #7012	5 µl
Phosphate Buffered Saline (PBS) #9872	995 µl

1. Weigh 1 mg fresh tissues for each reaction.
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.
3. Immediately transfer minced tissue to 1 ml of Fixation Buffer 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 the amount of Fixation Buffer used in Step 3 and Fixation Wash Buffer used in Step 7 accordingly.

4. Incubate at room temperature for 2 min.
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 supernatant.
7. Resuspend tissue with 1 ml of Fixation Wash Buffer.
8. Centrifuge for 5 min at 2,000 x g at 4°C and remove the supernatant 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 Complete Wash Buffer 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 Complete Wash Buffer.
13. Centrifuge cell suspension for 3 min at 3,000 x g at room temperature and remove the supernatant.
14. Wash the cell pellet a second time by repeating steps 12 and 13 one time.
15. For each reaction, add 100 µl of Complete Wash Buffer and resuspend the cell pellet by gently pipetting up and down.
16. Immediately proceed to the CUT&Tag Protocol, Section III.

C. Determination of Cell Sensitivity to Digitonin

In the CUT&Tag protocol, the addition of digitonin to the buffers facilitates the permeabilization of cell membranes and entry of the primary antibody, secondary antibody, and pAG-Tn5 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 exhibit varying 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:

• Warm Digitonin Solution #16359 at 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 during use. Store at -20°C when finished for the day.
• Prepare 100 µl of 1X Wash Buffer per reaction. It is not necessary to add spermidine or Protease Inhibitors in the buffer for this test.

1X Wash Buffer	Volume (per reaction)
10X Wash Buffer (CUT&RUN, CUT&Tag) #31415	10 µl
Nuclease-free Water #12931	90 µl

1. In a 1.5 ml tube, collect 100,000 cells (from CUT&Tag Protocol Section II-A, Step 1), centrifuge for 3 min at 600 x g at room temperature and withdraw the supernatant. For tissue, collect disaggregated cells from 1 mg of tissue (from CUT&Tag Protocol Section II-B, Steps 1-8).
2. Resuspend cell pellet in 100 µl of 1X Wash Buffer.
3. Add 2.5 µl Digitonin Solution #16359 to each reaction 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 #16359 added to each reaction and repeat steps 1-5 until > 90% cells are permeabilized and stained. Use this amount of Digitonin Solution #16359 in CUT&Tag Protocol Sections I-V.

D. Troubleshooting Guide

Problem	Possible Causes	Recommendation
1. Concanavalin A beads clump during the experiment.	Some bead clumping is normal and is not usually deleterious to the assay.	Resuspend clumped beads by gently pipetting up and down. Resting tubes on a tube rack instead of rocking or rotating sample tubes may help to minimize beads clumping and drying on tube walls.
	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 (CUT&Tag Protocol Section III, Step 2).
	Cells are lysing during preparation.	Be sure to prepare live cells at room temperature and as quickly as possible to minimize cell stress (CUT&Tag Protocol Section II).
	Digitonin concentration may be too high.	Some cells may be more sensitive to digitonin and lyse at higher concentrations. Reduce the amount of digitonin to a sufficient level for cell permeabilization (see CUT&Tag Protocol APPENDIX A).
2. Low yield, low sequencing depth, or no signal in NG-seq analysis.	Cell count is inaccurate, cells are lost or lysing during preparation.	Starting cell culture should be 60-90% confluent and look healthy (> 90% live cells). Make sure to get an accurate cell count using an automated cell counter or hemocytometer.
		Do not treat cells with trypsin for longer than necessary, as this may interfere with ConA bead binding.
		Be sure to prepare cells at room temperature and as quickly as possible to minimize cell stress.
		Perform initial cell wash steps in one vial to minimize cell loss (CUT&Tag Protocol Section II).
	Cells are over-fixed.	We recommend using live cells or fresh tissues for CUT&Tag assay whenever possible. If live cells or fresh tissues are not available, then we recommend lightly fixing the cells/tissues for 2 minutes in 0.1% formaldehyde. Over-fixation can lead to inhibition of the tagmentation reaction and loss of signal.
	Too few cells are used.	Use 100,000 cells per reaction whenever possible. Histone modifications may work with as few as 5,000 cells and transcription factors and cofactors may work with as few as 20,000 cells. However, the number of cells required to get good enrichment is highly dependent on the abundance of the target protein and avidity of the antibody. If no enrichment is seen when using fewer than 100,000 cells, then increase the number of cells per reaction. Use 100,000 cells per reaction whenever possible.
	If starting with low cell number, too much cell media is in the reaction for Concanavalin A beads binding.	During Concanavalin A Bead binding, having greater than 40% of cell culture media in the reaction reduces cell binding with beads and results in cell loss. Spin cell suspension to remove medium so that less than 40 µl per reaction is left. Then add Complete Wash Buffer to a total of 100 µl reaction for optimal Concanavalin A Beads binding.
	Tissue samples are not completely disaggregated.	Disaggregate tissue samples into single cell suspension, where no tissue chunks are observed. For fibrous tissue types that are difficult to be completely disaggregate, use more starting amount of tissue to compensate for the cell loss during tissue disaggregation.
	Digitonin is not effectively permeabilizing the cells.	Ensure Digitonin Solution #16359 is completely thawed and in solution before use. Occasionally digitonin crashes out of solution after placed on ice for hours.
		Be sure to test and confirm that the amount of digitonin used is sufficient to permeabilize your specific cell line (see CUT&Tag Protocol APPENDIX C).
		Place Digitonin Solution #16359 on ice when using and store it at -20°C when finished for the day. The Digitonin Solution must be stored at -20°C when not in use.
	pAG-Tn5 enzyme is not working properly in the assay.	The shelf life of pAG-Tn5 is 6 months. Do not use expired pAG-Tn5.
		The pAG-Tn5 requires Mg2+ divalent cations for activity. Be sure to add appropriate amount of magnesium chloride in the Tagmentation Buffer for enzyme activation (CUT&Tag Protocol Section V, Step 12).
		Be sure to add enough pAG-Tn5 (2 µl) to each CUT&Tag reaction and incubate at 37°C for 1 hr to allow sufficient tagmentation of chromatin (CUT&Tag Protocol Section V, Step 13).
	Antibody does not work in the CUT&Tag assay.	Not all antibodies work in CUT&Tag, especially antibodies against non-histone targets. If possible, use CUT&Tag validated antibodies.
		Be sure to include Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb #9751 as a positive control to confirm the overall experiment worked.
	Not enough PCR cycles used during library preparation.	Depending on starting cell numbers per reaction, use 13-16 PCR cycles to amplify tagmented DNA. Please refer to the protocol for CUT&Tag Dual Index Primers and PCR Master Mix for Illumina #47415.
3. NG-seq exhibits signal in unpredicted genome regions.	Non-specific pAG-Tn5 tagmentation occurs across the genome.	Do not over-dilute the 10X High Salt Digitonin Buffer used during the tagmentation reaction (CUT&Tag Protocol Section V, Step 12). The high salt concentration is critical to preventing non-specific binding of pAG-Tn5 to open chromatin regions.
		Use CUT&Tag validated antibodies to guide pAG-Tn5 to specific gene loci.
		Do not use an excessive starting amount of cells or tissues in one reaction.
		Non-specific pAG-Tn5 binding leads to increased enrichment of open chromatin, as seen with ATAC-seq assays. The positive control Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb #9751, which enriches for open and active gene promoters, shows significant overlap with ATAC-seq enrichment. Comparison of the target-specific antibody enrichment with that of the tri-methyl-histone H3 antibody can be used to determine the extent of non-specific pAG-Tn5 binding and tagmentation.