CUT&RUN kit protocol
- Section I: CUT&RUN buffer prep
- Section II: ConA bead activation
- Section III: Binding cells to activated beads
- Section IV: Antibody binding
- Section V: pAG-MNase binding
- Section VI: Targeted chromatin digestion and release
- Section VIIa: DNA purification for Kit Version 4
- Section VIIb: DNA purification for Kit Versions 1-3
CUT&RUN Protocol: Day 1
Section I: CUT&RUN buffer prep (~30 min)
- Buffers are prepared FRESH on Day 1 of every CUT&RUN experiment.
- 0.01% Digitonin is optimal for permeabilizing K562, MCF7, and A549 cells and is recommended for reactions using nuclei. For other cell types, Digitonin conditions MUST be optimized for efficient cell permeabilization. See this article for instructions.
- Volumes in Table 1 are per CUT&RUN reaction and include 20% excess to account for pipetting errors. You do NOT need to add additional volume.
- Gather kit reagents stored at 4˚C and -20˚C needed for Day 1: ConA beads, Bead Activation Buffer, Pre-Wash Buffer, Digitonin, Spermidine, H3K4me3 and IgG control antibodies, K-MetStat Panel. Place on ice to thaw/equilibrate.
- Dissolve 1 protease inhibitor tablet (Roche) in 2 mL water for a 25X Protease Inhibitor stock. After buffer prep, the remaining 25X stock can be stored for 12 weeks at -20˚C.
- Prepare Wash Buffer by combining Pre-Wash Buffer, 25X Protease Inhibitor, and 1 M Spermidine as outlined in Table 1. Store final buffer at RT.
- To a new tube labeled Cell Permeabilization Buffer, add Wash Buffer as outlined in Table 1. Add 5% Digitonin as optimzed for your cell type (see Important Notes on Buffer Prep, above)
- In a new 1.5 mL tube labeled Antibody Buffer, combine Cell Permeabilization Buffer and 0.5 M EDTA as described in Table 1. Place final buffer on ice.
- Store remaining Cell Permeabilization Buffer at 4˚C for use on Day 2.
Table 1. Buffer recipes for CUT&RUN. Includes extra volume to account for pipetting error.
Section II: ConA bead activation (~30 min)
Tips for working with magnetic ConA beads
- Do NOT use ConA beads that have been frozen and/or appear black, granular, or clumpy.
- Do NOT let ConA beads dry out. Avoid disturbing beads with pipette while on magnet.
- Keep activated ConA beads on ice and use within four hours of activation.
- Gently resuspend ConA beads and transfer 11 μL per reaction to a 1.5 mL tube.
- Place tube on a magnet, allow slurry to clear. Pipette to remove supernatant.
- Remove tube from magnet. Immediately add 100 μL per reaction cold Bead Activation Buffer and pipette to resuspend. Return tube to magnet, allow slurry to clear, and pipette to remove supernatant.
- Repeat the previous step one time.
- Resuspend beads in 11 μL per reaction cold Bead Activation Buffer.
- Aliquot 10 μL per reaction of bead slurry into 8-strip tubes. Place on ice.
Section III: Binding cells to activated beads (~30 min)
Guidelines for successful sample prep
- High quality sample prep is essential to CUT&RUN success and is the main variable we see when troubleshooting customer experiments.
- Freshly isolated, native (i.e. unfixed) cells are the preferred input for CUT&RUN. For sample inputs other than native suspension cells (e.g. adherent cells) see Sample Prep.
- Count and examine cells using Trypan Blue staining. Check cellular morphology, integrity, and viability at the three steps outlined below. For more, see this article.
- Initial cell harvest. Cells have high viability and expected morphology. Acceptable viability is dependent on cell type and experimental conditions.
- Before ConA bead binding. Cells in CUT&RUN Wash Buffer have expected morphology and minimal lysis and/or sample loss.
- After binding to ConA beads. The supernatant contains very few cells and the sample shows that all cells are bound to ConA beads.
- Count starting cells and confirm integrity using Trypan Blue staining. Harvest 500,000 cells per reaction (plus 10% excess) and spin at 600 x g for 3 min at room temperature (RT). Remove supernatant.
- Resuspend cells in 100 μL per reaction RT Wash Buffer by gentle yet thorough pipetting. Spin at 600 x g, 3 min, RT. Pipette to remove supernatant.
- Repeat the previous step one time.
- Resuspend cells in 105 μL per reaction RT Wash Buffer. Count and examine integrity of prepared cells using Trypan Blue staining.
- Add 100 µL cells to 10 µL ConA beads in 8-strip tubes. Gently vortex to mix and quick spin in a mini-centrifuge to collect slurry (beads should not settle).
- Incubate bead-cell slurry for 10 min at RT. Cells will adsorb to beads.
- During incubation, retrieve a compatible 8-strip tube magnetic rack. In addition, if using a multi-channel pipettor (recommended), place a multi-channel reagent reservoir on ice. Fill with cold Antibody Buffer.
- If using the EpiCypher 8-strip tube magnet, use the high-volume side unless otherwise noted.
- After the 10 min incubation, place tubes on a magnet and allow slurry to clear. If bead binding was successful, the supernatant should not contain cells. To confirm, save 10 μL supernatant to assess sample integrity using Trypan Blue staining.
- Pipette to remove and discard the remaining supernatant. Remove tubes from magnet and immediately add 50 μL cold Antibody Buffer to each reaction.
- Pipette to resuspend. Confirm ConA bead binding.
Section IV: Antibody binding (~30 min + overnight)
Antibody Incubation Notes
- Add K-MetStat Panel to control reactions BEFORE adding the primary antibody.
- Do NOT rotate or invert tubes. Rotation causes ConA beads to stick to tube sides and dry out, reducing yields. Use a nutator for incubations and elevate tube caps as suggested.
- Quick spin the K-MetStat Panel and pipette to resuspend - do NOT vortex. Add 2 µL K-MetStat Panel to reactions designated for H3K4me3 positive and IgG negative control antibodies. Gently vortex to mix and quick spin tubes. If using fewer than 500,000 cells, decrease K-MetStat Panel amount as in Table 2 (below).
- Add 0.5 μg primary antibody (or manufacturer's recommendation) to each reaction. For positive and negative control reactions, add 1μL H3K4me3 Positive Control Antibody and 1μL IgG Negative Control Antibody, respectively. For antibodies stored in viscous glycerol solutions, ensure accurate pipetting: aspirate slowly, check tip for accuracy, and pipette up and down to clear the solution from tip.
- Gently vortex reactions to mix. Incubate overnight on a nutator at 4˚C with tube caps elevated (Figure 1). Do NOT rotate - see Antibody Incubation Notes, above.
Figure 1. 8-strip tubes placed on a nutator at a 45 degree angle with caps elevated.
|Cell number||Panel dilution||Volume added to reaction|
|500,000||Use stock||2 μL|
|50,000 or fewer||1:10||2 μL|
Table 2. Scale the amount of K-MetStat Panel to the number of cells. For <500,000 cells, prepare a working stock dilution of the K-MetStat Panel in Antibody Buffer the day of experiment.
CUT&RUN Protocol: Day 2
Section V: pAG-MNase binding (~40 min)
Notes on ConA bead-cell clumping
- It is essential that ConA beads remain in solution during pAG-MNase binding and digestion. Excessive bead clumping leads to sample loss, reduces yields, and negatively impacts quality.
Guidelines for high-throughput processing
- Multi-channel pipetting is recommended to improve reliability and experimental throughput. To easily dispense buffers, use multi-channel reagent reservoirs (keep on ice).
- For 8-strip tubes, remove and replace buffers one strip at a time to avoid ConA bead dry out.
- If using a multi-channel pipettor (recommended), place a multi-channel reagent reservoir on ice. Fill with cold Cell Permeabilization Buffer.
- Remove tubes from 4˚C incubation and quick spin to collect liquid. Note that beads may settle overnight (Figure 2, below), but this will not impact results.
- Place tubes on magnet and allow slurry to clear. Pipette to remove supernatant. If using a multi-channel pipettor, remove and replace buffers one tube strip at a time to avoid ConA bead dry-out.
- Keeping tubes on magnet, add 200 μL cold Cell Permeabilization Buffer to each reaction. Pipette to remove supernatant.
- Repeat the previous step one time (keep tubes on magnet).
- Remove tubes from magnet and immediately add 50 μL cold Cell Permeabilization Buffer to each reaction. Gently vortex to mix. Beads may become clumpy at this stage of the protocol, but can be dispersed by gentle pipetting. The end of a pipette tip can be cut off to help mix delicate cells.
- Add 2.5 μL pAG-MNase to each reaction. Gently vortex and/or pipette to thoroughly mix beads and evenly distribute enzyme.
- Incubate reactions for 10 min at RT.
- Quick spin tubes, place on magnet, and allow slurry to clear. Pipette to remove supernatant.
- Keeping tubes on magnet, add 200 μL cold Cell Permeabilization Buffer directly onto beads. Pipette to remove supernatant.
- Repeat one time.
- Remove tubes from magnet. Immediately add 50 μL cold Cell Permeabilization Buffer to each reaction. Gently vortex to mix and disperse clumps by pipetting.
Figure 2. Settling of ConA beads after overnight incubation at 4˚C.
Section VI: Targeted chromatin digestion and release (~3 hrs)
Guidelines for using E. coli spike-in DNA
- Reconstitute the lyophilized E. coli Spike-in DNA prior to first use: Quick spin tube before opening to collect E. coli DNA in bottom of tube. Add 200 μL DNase-free water and thoroughly vortex tube on all sides to resuspend E. coli DNA. Store at -20˚C.
- Aim for E. coli Spike-in DNA to comprise 0.5 - 5% (ideally ~1%) total sequencing reads.
- This protocol is optimized using 0.5 ng E. coli DNA and 500,000 cells per reaction. If using fewer than 500,000 cells per reaction, dilute E. coli Spike-in DNA as outlined here.
- Place tubes on ice. Add 1 μL 100 mM Calcium Chloride to each reaction. Gently vortex and/or pipette to evenly resuspend beads and ensure efficient digestion.
- Incubate tubes on nutator (capped ends elevated) for 2 hours at 4ºC.
- Retrieve E. coli Spike-in DNA. Reconstitute DNA prior to first use (see Guidelines, above) or thaw previously resuspended DNA on ice and quick spin before use.
- Prepare a Stop Master Mix in a 1.5 mL tube. Per reaction, combine 33 μL Stop Buffer and 1 μL E. coli Spike-in DNA (0.5 ng). Gently vortex to mix.
- At the end of the 2 hour incubation, quick spin 8-strip tubes to collect liquid. Add 34 μL Stop Master Mix to each reaction and gently vortex to mix.
- Place reactions in a thermocycler set to 37˚C. Incubate for 10 min.
- Transfer supernatants containing CUT&RUN-enriched DNA to new tubes:
Section VIIa: DNA purification (~30 min)
- Prepare 85% Ethanol (EtOH) FRESH using a 100% EtOH stock and molecular biology grade water. Make 500 μL per reaction: 425 μL 100% EtOH + 75 μL water. These calculations include extra volume to account for pipetting error.
- Retrieve SPRIselect reagent, manufactured by Beckman Coulter, Inc., from the kit RT reagents. Vortex thoroughly to resuspend.
- Slowly add 119 μL SPRIselect reagent (1.4X volume) to each reaction. Ensure pipette tip is free of extra droplets before dispensing beads to reactions.
- Gently vortex tubes to mix and quick spin to collect liquid in tube bottom. Incubate 5 min at RT.
- Place tubes on magnet for 2-5 min at RT. Pipette to remove supernatant, being careful not to disturb beads with pipette tip.
- Keeping tubes on magnet, add 180 μL 85% EtOH directly onto beads. Pipette to remove supernatant.
- Repeat the previous step one time.
- Remove tubes from magnet and quick spin, caps facing in. Beads should stay in place on side of tube. Return to magnet and pipette to remove residual EtOH.
- Remove tubes from magnet, leaving caps open. Air-dry beads for 2-3 min at RT, or until liquid has evaporated but beads still appear damp matte brown. If beads become crackly, they are too dry (Figure 3, below).
- Add 17 μL 0.1X TE Buffer to each reaction to elute DNA.
- Gently vortex tubes to resuspend beads and quick spin. Incubate 2 min at RT.
- Place tubes on magnet for 2 min at RT.
- Transfer 15 μL CUT&RUN-enriched DNA to new 8-strip tubes.
- Use 1 μL to quantify DNA using the Qubit fluorometer and 1X dsDNA HS Assay Kit.
Safe pause point.
Figure 3. SPRIselect reagent shown at various levels of dry. Ensure your ConA beads are not over or under-dry when eluting DNA.
Section VIIb: DNA purification for Kit Versions 1-3:
Notes before starting DNA purification
- Prior to first use, add 6.9 mL isopropanol to DNA Binding Buffer.
- Prior to first use, add 20 mL ≥95% ethanol to Wash Buffer.
- A vacuum manifold can be used in place of centrifugation. For each step, add the indicated buffer, turn the vacuum on, and allow the solution to pass through the column before turning the vacuum off.
- Add 420 μL DNA Binding Buffer to each reaction. Mix well by vortexing
- For every CUT&RUN reaction, place a DNA Cleanup Column into a DNA Collection Tube. Load each reaction onto a column and label the top.
- Spin at 16,000 x g, 30 sec, RT. Discard flow-through. Place column back into collection tube.
- Add 200 μL DNA Wash Buffer to each column. Spin at 16,000 x g, 30 sec, RT. Discard flow-through. Place column back into collection tube.
- Repeat previous step one time.
- Spin at 16,000 x g, 30 sec, RT to completely dry the column
- Carefully remove the column from the collection tube, ensuring it does not contact the flow-through. Transfer column to a clean pre-labeled 1.5 mL tube.
- Add 12 μL DNA Elution Buffer, taking care to ensure the buffer is added to the center of the column rather than the wall. Tap the column & microfuge collection tube on the benchtop to ensure all droplets are absorbed onto the resin.
- DNA can be eluted in 6–20 μL; 12 µL is recommended. Larger elution volumes, longer incubation times, and/or multiple rounds of elution may improve DNA yield. However, DNA concentration will be reduced with larger elution volume.
- Let sit 5 minutes, then spin at 16,000 x g, 1 min, RT.
- Vortex eluted material to mix. Use 1 μL to quantify the CUT&RUN DNA using the Qubit fluorometer with the 1X dsDNA HS Assay Kit per the manufacturer's instructions.
Safe pause point.
Confirming CUT&RUN success
- There is no typical yield for CUT&RUN, as results vary by cell type, number, target, and antibody performance.
- In general, yields from H3K4me3 positive control are slightly greater than IgG negative control. However, if yields are similar, this does NOT imply assay failure. Read more here.
- We recommend aiming for ≥5 ng CUT&RUN enriched DNA, which will enable robust library prep. See this article for troubleshooting low yields.
- Electrophoretic analysis (e.g. TapeStation) and/or qPCR of CUT&RUN DNA at this step is NOT recommended.
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