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CUT&RUN Troubleshooting Guide

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.

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.

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.
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.
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