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CUT&RUN Troubleshooting Guide

A: Determination of Cell Sensitivity to Digitonin

In the CUT&RUN kit 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 at 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.

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 (+ spermidine + PIC) 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 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.

C: Troubleshooting Guide

Problem Possible Causes Recommendation
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. Rocking instead of rotating sample tubes may help with 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 (Section II, Step 7).
Cells are lysing during preparation. Be sure to prepare live cells at room temperature and as quickly as possible to minimize cell stress (Section I).
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 extremely low cell numbers (≤20,000 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® due to the low starting number.
Cell count is off, cells are lost or lysed 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.
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).
Cells are over-fixed. When choosing to fix cells, light fixation (0.1% formaldehyde for 2 min) is strongly recommended. For some difficult tissue types (fibrous tissues), medium fixation conditions (0.1% formaldehyde for 10 min) can be used if optimal results are not observed with light fixation.
Too few cells are used. Use 100,000 cells per reaction whenever possible. A minimum of 5,000 cells are required for histone modifications. For transcription factors and cofactors, at least 10,000 cells are needed.
Too much medium is in the reaction for Conconavalin A beads binding. With over 40% of medium in the reaction, Conconavalin A Beads binding with cells is greatly reduced and cells may be lost. Spin cell suspension to remove medium so that less than 40 µl per reaction is left. Then add 1X 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 until no tissue chunks are observed. For fibrous tissue types that are difficult to be completely disaggregate, increase your starting amount of tissue to compensate for cell loss during tissue disaggregation.
Digitonin is not effectively permeabilizing the cells. Be sure to store Digitonin Solution #16359 at -20°C when not in use, as it is less stable 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 at 20°C.
The pAG-MNase requires Ca2+ divalent cations for activity. Be sure to add calcium chloride for activation of the enzyme (Section IV, Step 8).
Be sure to digest for 30 min to allow the enzyme to sufficiently digest the chromatin (Section IV, Step 9).
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 #9751 to show your assay is working.
3. No signal in qPCR or NG-seq analysis. Review all possible causes in question 2. See all reccommendations for question 2.
Digestion conditions may be too cold. We have found that performing the digestion on ice (0°C) can significantly decrease the recovery of targeted chromatin fragments, resulting in decreased qPCR and NG-seq signals. Please be sure to perform the digestion at 4°C on a cooling block or in a refrigerator.
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.
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. Use as much of the starting DNA as possible and up to 20 PCR-amplification cycles.
Due to the low starting amount of DNA, modifications to a standard NGS-library prep protocol are necessary. Please be sure to follow the recommendations for CUT&RUN DNA library preparation, which is different than that for ChIP DNA library preparation (See Section VIII of CUT&RUN Assay Kit #86652).
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 (Section I).
Digestion conditions may be too warm. Digestion should be performed at 4°C in a cooling block or refrigerator. Digestion at higher temperatures can significantly increase background signal.
Make sure to pre-cool samples and keep calcium chloride on ice prior to initiating the digest.
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 IV, 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.