CUT&RUN Troubleshooting Guide

Select the CUT&RUN Problem you are EncounteringConcanavalin A beads clump during the experimentNo DNA is detected in the purified DNA samples using a picogreen-based DNA quantification assayNo signal in qPCR or NG-seq analysisHigh background signal in qPCR or NG-seq analysis

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