CUT&RUN-seq protocol
Licia Miller Product Manager
Targets and Release Using Nuclease (CUT&RUN) experimental technology is an emerging chromatin analysis method for studying protein-DNA interactions. It uses antibodies to specifically label target proteins, and then uses nucleases bound to antibodies (such as pAG-MNase) to cut DNA fragments that interact with these proteins. These DNA fragments are then purified by immunoprecipitation technology and sequenced for analysis, thereby accurately locating the binding sites of proteins on the genome.
ChIP and CUT&RUN are both techniques used to study protein-DNA interactions, but they differ in the methods and experimental procedures for DNA fragmentation. ChIP usually relies on physical methods (such as ultrasound) to randomly fragment the chromatin of fixed cells, while CUT&RUN uses antibody-guided nucleases to directly cut DNA near the target protein in living cells or slightly fixed cells, thereby achieving more precise DNA fragmentation positioning and lower background noise.
Compared with ChIP technology, CUT&RUN provides a more accurate and efficient method for chromatin-protein interaction analysis, which can reduce nonspecific binding and improve the signal-to-noise ratio of the experiment.
Stage 1 Cell harvesting and magnetic bead preparation
Experimental Steps
1. Collect 5,000 to 100,000 cells per antibody/MNase reaction and an additional 5,000 to 100,000 cells for the input sample if input is required . Be sure to include reactions for positive and negative controls.
Note: For adherent cell lines, cells must first be trypsinized and neutralized with at least 3 volumes of culture medium. To preserve the integrity of the cell morphology, scraping cells from the culture dish is not recommended.
2. Count the cells using a hemocytometer or other cell counter and take the required number of cells for each reaction. For HeLa cells, use 2.5 × 105 cells per reaction.
3. Add 2.7 µl of 37% formaldehyde per 1 ml of cell suspension to achieve a final concentration of 0.1%. Vortex to mix and incubate at room temperature for 2 minutes.
4. Add 100 µl of glycine solution (10×) per 1 ml of fixed cell suspension to stop crosslinking. Vortex to mix and incubate at room temperature for 5 minutes.
5. Centrifuge the cell suspension at 3,000 × g for 3 minutes at 4°C and remove the liquid, and proceed immediately to the next step. Alternatively, the fixed cell pellet can be stored at -80°C for up to 6 months before use.
Note: When total cell numbers are < 100,000, the cell pellet after centrifugation may not be apparent to the naked eye, and it is easy to lose cells during the wash steps. Therefore, when working with low cell numbers, we recommend skipping the wash steps below.
6. Prepare wash buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine) and add protease inhibitor cocktail before use.
7. Add 1 mL of wash buffer to the cells, spin the cells at 600 × g for 3 minutes, remove the supernatant and repeat the wash 1-3 times (depending on the number of cells; if the cell pellet is not visible to the naked eye, the number of washes can be reduced).
NOTE: For the input sample, add 100 µl of 1× Wash Buffer at this time and gently pipette up and down to resuspend the cell pellet. Transfer 100 µl of cells to a new tube and store at 4°C as the input sample.
8. Prepare binding buffer (20 mM HEPES pH 7.5, 10 mM KCl, 1 mM CaCl2, 1 mM MnCl2), wash the ConA beads with 1 mL of binding buffer on a magnetic stand, and resuspend the beads with binding buffer to prepare ConA bead slurry. 10 µL of beads are required for each reaction.
Avoid vortexing the ConA bead suspension when resuspending the beads, as repeated vortexing may displace ConA from the beads.
9. Mix the cells and magnetic beads at room temperature for 20 minutes, mixing gently every 4 minutes to allow the cells to bind to the activated magnetic beads.
10. Separate the cell-bound beads from the solution by placing the tube on a magnetic stand for 1-2 minutes until the solution becomes clear and discard the supernatant and retain the beads.
Stage 2 Antibody Binding
Required Materials
- Wash buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine, protease inhibitor cocktail)
- 0.5 M EDTA stock solution
- 5% Digitonin stock solution
- Antibody Buffer
- Antibodies validated for CUT&RUN
- Horizontal rotary shaker
- Magnetic rack
Experimental Steps
1. Prepare antibody buffer. Take an appropriate amount of washing buffer (containing protease inhibitors) and add EDTA and Digitonin stock solution to make the final concentration reach 0.2 mM EDTA and 0.05% Digitonin.
2. Resuspend the beads in Antibody Buffer. If preparing multiple reactions in the same tube, resuspend approximately 100 µL per reaction and aliquot into separate tubes.
3. Add the antibody to the magnetic bead mixture at the recommended dilution and incubate overnight at 4°C on a horizontal shaker, taking care to prevent the ConA beads from drying out.
Stage 3 pAG-MNase binding
Required Materials
- Wash buffer (20 mM HEPES, 150 mM NaCl, 0.5 mM spermidine, protease inhibitor cocktail)
- 5% Digitonin stock solution
- Digitonin Wash Buffer ( containing 0.05% Digitonin)
- Horizontal rotary shaker
- Magnetic rack
Experimental Steps
1. Place the tube on a magnetic stand for 1-2 minutes until the solution becomes clear, discard the supernatant and retain the magnetic beads to separate the cell-bound magnetic beads from the solution.
2. Prepare Digitonin Wash Buffer by diluting the digitonin stock solution in Wash Buffer (containing a protease inhibitor cocktail) to a final concentration of 0.05%.
3. Add 200 µL to each reaction Wash the beads with Digitonin Wash Buffer, place the tube on a magnetic stand for 1 minute until the solution is clear, discard the supernatant and keep the beads . Repeat the wash once.
4. Use 50 µL Resuspend the magnetic beads in Digitonin wash buffer, add pAG-MNase to a final concentration of 700 ng/μL, place the magnetic beads and pAG-MNase solution on a horizontal shaker at 130 rpm and incubate at room temperature for 1-2 hours.
Stage 4 DNA Digestion
Required Materials
- Low salt buffer (20 mM HEPES pH 7.5, 0.05% Digitonin, 0.5 mM Spermidine)
- Incubation buffer (3.5 mM HEPES pH 7.5, 10 mM CaCl2, 0.05% Digitonin)
- Magnetic rack
Experimental Steps
1. Place the tube on a magnetic stand for 1-2 minutes until the solution becomes clear, discard the supernatant and retain the magnetic beads to separate the cell-bound magnetic beads from the solution.
2. Prepare low salt buffer and incubation buffer, add Digitonin before use. Place the buffer on ice after preparation.
3. Add 200 µL to each reaction Digitonin Wash Buffer, incubate for 5 minutes, place the tube on a magnetic stand for 1 minute until the solution becomes clear, discard the supernatant and retain the beads. Repeat the wash once.
4. Wash the beads with low salt buffer. Add 200 µL to each reaction. Low salt buffer, incubate for 5 minutes, place the tube on a magnetic stand for 1 minute until the solution becomes clear, discard the supernatant and retain the magnetic beads.
5. Add 100 µL of incubation buffer and incubate on ice for 15 minutes to digest chromatin, mixing occasionally. Place the tube on a magnetic stand for 1 minute until the solution is clear, discard the supernatant and retain the beads.
Stage 5 DNA elution and sequencing
Required Materials
- Stop buffer (20 mM EGTA, 170 mM NaCl, 0.05% Digitonin, 50 µg/mL RNAse A, 25 µg/mL glycogen)
- Thermomixer
- Magnetic rack
Experimental Steps
1. Add 100 µL of ice-cold stop buffer and place the beads in a thermomixer, shake at 700 rpm, and incubate at 37°C for 30 minutes to stop the reaction and release the digested DNA fragments into the solution.
2. Place the tube on the magnetic rack for 1 minute until the solution becomes clear, collect the supernatant and transfer it to a new tube. The supernatant now contains DNA fragments.
3. Extract DNA using a commercial kit and prepare for sequencing.
4. Send the DNA fragments for sequencing.
For more product details, please visit Aladdin Scientific website.