Bench top CUT&RUN with antibodies-online™ CUT&RUN Sets
Antibodies Online Gmbh
Abstract
CUT&RUN (Cleavage Under Targets and Release Using Nuclease) offers a novel approach
to pursue epigenetics. The method is designed to map genome wide transcription factor
binding sites, chromatin-associated complexes, and histone variants and post-translational
modifications.
Here we provide an updated CUT&RUN protocol that incorporates two variants, one is optimized to further reduce background noise which especially helps when working with low cell numbers and abundant antigens.
In CUT&RUN is performed in situ on immobilized, intact cells without crosslinking. DNA fragmentation is achieved using micrococcal nuclease that is fused to Protein A and/or Protein G (pA/G-MNase). The fusion protein is directed to the desired target through binding of the Protein A/G moiety to the Fc region of an antibody bound to the target. DNA under the target is subsequently cleaved and released and the pA/G-MNase-antibody- chromatin complex is free to diffuse out of the cell. DNA cleavage products are extracted and then processed by next generation sequencing (NGS).
All steps from live cells to sequencing-ready libraries can be performed in a single tube on the benchtop or a microwell in a high-throughput pipeline, and the entire procedure can be performed in one day.
Before start
General remarks
• The original CUT&RUN protocol in Skene et al. (2018)(Skene, PJ; Henikoff JG;
Henikoff, 2018) recommends sample sizes of 100 to 1000 mammalian cells
for abundant antigens such as H3K27me3 or CTCF. This protocol adapted
from Meers et al. (2019)(Meers, Bryson, et al., 2019) is suitable for up to
500.000 cells.
This protocol is intended to give a general outline of the CUT&RUN protocol.
It has to be adjusted according to the:
» Cell type. Your specific cell type might necessitate different treatments
prior to the CUT&RUN procedure, e.g. disintegration of tissue, generation of
spheroblasts,
» MNase digestion time points during the optimization.
Different samples, approaches, and digestion time points are uniformly
referred to in the protocol as “samples”.
• To minimize DNA breakage during sample preparation, avoid cavitation
through vigorous resuspension and vigorous vortexing.
• Keep cells at room temperature during all steps prior to the addition of anti-
body to minimize stress on the cells and DNA breakage.
• All steps from the incubation with the primary antibodies on should be car-
ried out at 4°C.
Antibody selection
An antibody specific for your protein of interest is crucial to direct the pA/G-
MNase mediated nucleic acid cleavage to the intended site. The Protein A/G
portion tethers the fusion protein to the Fc region of the antibody bound to its
antigen. This allows the pA/G-Mnase nuclease portion to cleave the nucleic
acid under the targeted protein and to release the nucleic acid.
Validated CUT&RUN antibodies - available at antibodies-online
Depending on the host species and isotype of the antibody and the Protein
A and/or Protein G MNase fusion protein, it can be necessary to include a
secondary antibody for pA/G-MNase binding (Skene & Henikoff, 2017). If the
pA-MNase is used in conjunction e.g. with a primary mouse IgG1 or goat IgG
antibody it is recommended to use a rabbit secondary antibody (Section V).
Protein A binds well to rabbit or guinea pig IgG antibodies but only poorly to
mouse IgG1 or goat IgG. No additional secondary antibody is needed when
using pA/G-MNase (Meers, Bryson, et al., 2019).
The positive Control H3K27me3 antibody (ABIN6923144) and Negative
Control Guinea Pig anti-Rabbit IgG antibody (ABIN6923140) are important
to assess cleavage and chromatin release without the need to sequence the
released DNA fragments. Do not use a no-antibody negative control: untethe-
red pA/G-MNase will unspecifically bind and cleave any accessible DNA, thus
increasing background signal.
Steps
REAGENT SETUP (for 12 samples)
» Wash buffer (165 mL)
| A | B | C |
|---|---|---|
| Component | Volume | Final concentration |
| ddH2O | 156.7 mL | - |
| 1 M HEPES pH 7.5 | 3.3 mL | 20 mM |
| 5 M NaCl | 4.95 mL | 150 mM |
| 2 M Spermidine | 41.25 µL | 0.5 mM |
• Store Wash Buffer without protease inhibitors for up to one week at 4 °C.• Add protease inhibitors fresh before use, e.g.:1.65 mL Protease Inhibitor (EDTA-free)
» Binding Buffer (45 mL)
| A | B | C |
|---|---|---|
| Component | Volume | Final concentration |
| ddH2O | 43.6 mL | - |
| 1 M HEPES pH 7.5 | 900 µL | 20 mM |
| 1 M KCl | 450 µL | 10 mM |
| 1 M CaCl2 | 45 µL | 1 mM |
| 2.5 M MnCl2 | 16 µL | 1 mM |
• Store Binding Buffer for up to six months at 4 °C.
» Digitonin Wash Buffer (82.5 mL)
| A | B | C |
|---|---|---|
| Component | Volume | Final concentration |
| 1.2 % Digitonin | 3354 µL | 0.05 % |
| Wash Buffer | 79 mL | - |
• Store Digitonin Wash Buffer for up to one day at 4 °C.• Recommended Digitonin concentration ranges from 0.025% to 0.1%. • The effectiveness of Digitonin varies between batches. Test cell permeability using Trypan Blue to determine the optimal concentration to use.
» Antibody Buffer (1.5 mL)
| A | B | C |
|---|---|---|
| Component | Volume | Final concentration |
| 0.5 M EDTA | 6 µL | 2 mM |
| 10 % BSA | 15 µL | 0.1 % |
| Digitonin Wash Buffer | 1.5 mL | - |
• Store Antibody Buffer for up to one day at 4 °C until use.
» Low Salt Rinse Buffer (27 mL)
| A | B | C |
|---|---|---|
| Component | Volume | Final concentration |
| ddH2O | 25.3 mL | - |
| 1 M HEPES pH 7.5 | 540 µL | 20 mM |
| 2 M Spermidine | 6.75 µL | 0.5 mM |
| 1.2% Digitonin | 1125 µL | 0.05 % |
• Store Low Salt Rinse Buffer for up to one week at 4 °C until use.
» Low Salt Incubation Buffer (3 mL)
| A | B | C |
|---|---|---|
| Component | Volume | Final concentration |
| ddH2O | 2.8 mL | - |
| 1 M HEPES pH 7.5 | 10.5 µL | 3.5 mM |
| 1 M CaCl2 | 30 µL | 10 mM |
| 1.2% Digitonin | 125 µL | 0.05% |
• Store Low Salt Incubation Buffer for up to one week at 4 °C until use.
» Low Salt Stop Buffer (3 mL)
| A | B | C |
|---|---|---|
| Component | Volume | Final concentration |
| ddH2O | 2.8 mL | - |
| 5 M NaCl | 102 µL | 170 mM |
| 0.2 M EGTA | 300 µL | 20 mM |
| Store Low Salt Stop Buffer at 4 °C until use. | ||
| Add fresh before use: | ||
| 1.2 % Digitonin | 125 µL | 0.05% |
| RNase A (10 mg/mL) | 15 µL | 50 µg/mL |
| Glycogen (20 mg/mL) | 7.5 µL | 25 µg/mL |
| Optional: | ||
| heterologous spike-in DNA | - | 100 pg/mL |
I.Cell Harvest – at room temperature
Harvest 10,000 to 500,000 cells for each sample at Room temperature. Keep cells for each sample in separate tubes.
Centrifuge cell solution0h 3m 0s at 600x g at Room temperature. Remove the liquid carefully.
Gently resuspend cells in 1mL Wash Buffer by pipetting and transfer cell solution to a 1.5mL
microcentrifuge tube.
Centrifuge cell solution 0h 3m 0s at 600x g at Room temperature and discard the supernatant.
Repeat steps 4-5 thrice for a total of four washes.
Resuspend cell pellet for each sample in Wash Buffer 1mL Wash Buffer by gently pipetting.
II. Concanavalin A beads preparation
Prepare one 1.5mL microcentrifuge tube for each sample.
Gently resuspend the
Pipette 10µL CUT&RUN Concanavalin A Beads slurry for each sample into the 1.5mL microcentrifuge tubes.
Place the tubes on a magnet stand until the fluid is clear. Remove the liquid carefully.
Remove the microcentrifuge tube from the magnet stand.
Pipette1mL Binding Buffer into each tube and resuspend CUT&RUN Concanavalin A Beads by gentle pipetting.
Spin down the liquid from the lid with a quick pulse in a table-top centrifuge (max 100x g).
Place the tubes on a magnet stand until the fluid is clear. Remove the liquid carefully.
Remove the microcentrifuge tube from the magnet stand.
Repeat steps 13-16 twice for a total of three washes.
Gently resuspend the CUT&RUN Concanavalin A Beads in a volume of Binding Buffer corresponding to the original volume of bead slurry, i.e. 10µL per sample.
III. Cell immobilization – binding to Concanavalin A beads
Carefully vortex the cell suspension from step and add 10µL of the CUT&RUN Concanavalin A Beads in Binding Buffer prepared in section II to each sample.
Close tubes tightly and rotate for 0h 5m 0s to 0h 10m 0s at Room temperature.
IV. Cell permeabilization and primary antibody binding
Place the microcentrifuge tubes on a magnet stand until the fluid is clear.
Remove the liquid carefully.
Remove the microcentrifuge tubes from the magnet stand.
Place each tube at a low angle on the vortex mixer set to a low speed (approximately 1100rpm) and add 100µL Antibody Buffer containing digitonin .
Gently vortex the microcentrifuge tubes until the beads are resuspended.
For the positive control, add 5µL CUT&Tag rabbit anti-H3K4me3 IgG Positive
Control corresponding to a 1:20 dilution to the corresponding tube.
For the negative control, add 5µL CUT&RUN guinea pig anti-rabbit IgG Negative
Contro l corresponding to a 1:20 dilution to the corresponding tube.
In case you are using one of the CUT&RUN anti-DYKDDDDK antibodies ,
add 5µL corresponding to a 1:20 dilution to the corresponding tube.
For the remaining samples, add 1µL primary rabbit antibody - against your
protein of interest corresponding to a 1:100 dilution (or a volume corresponding
to the manufacturer’s recommended dilution for immunofluorescence).
Rotate the microcentrifuge tubes for 0h 5m 0s to 0h 10m 0s at 4Room temperature or 2h 0m 0s to at 4°C.
Spin down the liquid and place the tubes on a magnet stand until the fluid is clear. Remove the liquid carefully.
Remove the microcentrifuge tubes from the magnet stand.
Resuspend with 1mL Digitonin Wash Buffer and mix by inversion. If clumping occurs, gently remove the clumps with a 1mL pipette tip.
Repeat steps 30-32 once for a total of two washes.
V. Secondary antibody binding (optional)
Place the tubes on a magnet stand until the fluid is clear. Remove the liquid carefully.
Remove the microcentrifuge tubes from the magnet stand.
Vortex the sample at low speed (approximately 1100rpm) and add 100µL Digitonin Wash Buffer per sample along the side of the tube.
Tap to remove the remaining beads from the tube side.
Add 1µL
corresponding to a 1:100 dilution (or a volume corresponding to the manufacturer’s recommended dilution for immunofluorescence).
Rotate the microcentrifuge tubes for 1h 0m 0s at 4°C.
Spin down the liquid and place the tubes on a magnet stand until the fluid is clear. Remove the liquid carefully.
Remove the microcentrifuge tubes from the magnet stand.
Resuspend with 1mL Digitonin Wash Buffe r and mix by inversion. If clumping occurs, gently remove the clumps with a 1 mL pipette tip.
Repeat steps40-42 once for a total of two washes.
VI. Protein A-MNase or Protein AG-MNase Binding
Place the tubes on a magnet stand until the fluid is clear. Remove the liquid
carefully.
Remove the microcentrifuge tubes from the magnetic stand.
Vortex the sample at low speed (approximately 1100rpm) and add 50µL Digitonin Wash Buffer per sample along the side of the tube. Add 2.5µL
Alternatively:
Vortex the sample at low speed (approximately 1100rpm) and add 150µL Digitonin Wash Buffer containing 700ng/mL of your own pA/G-MNase preparation per sample along the side of the tube.
Rotate the microcentrifuge tubes for 1h 0m 0s at 4°C.
Spin down the liquid and place the tubes on a magnet stand until the fluid is clear. Remove the liquid carefully.
Remove the microcentrifuge tubes from the magnetic stand.
Resuspend with 1mL Digitonin Wash Buffer and mix by inversion. If clumping occurs, gently remove the clumps with a 1 ml pipette tip.
Repeat steps 48-50 once for a total of two washes.
VII. MNase digestion and release of pA/G bound chromatin fragments
Spin down the liquid from the lid with a quick pulse in a table-top centrifuge
(max 100x g,0h 0m 0s).
Place the tubes on a magnet stand until the fluid is clear. Remove the liquid carefully.
Resuspend with 1mL Low Salt Rinse Buffer and mix by inversion. If clumping
occurs, gently remove the clumps with a 1 mL pipette tip.
Spin down the liquid from the lid with a quick pulse in a table-top centrifuge
(max 100x g,0h 0m 0s).
Place the tubes on a magnet stand until the fluid is clear. Remove the liquid carefully.
Repeat steps 54-56 once for a total of two washes.
Place each tube at a low angle on the vortex mixer set to a low speed (approx. 1100rpm ) and add 200µL ice cold Low Salt Incubation Buffer per sample along the side of the tube.
Incubate tubes at 0°C for the desired time (default is 0h 30m 0s).
Place the tubes on a cold magnet stand until the fluid is clear. Remove the liquid carefully.
Remove the microcentrifuge tubes from the magnet stand.
Resuspend with 200µL Low Salt Stop Solution and mix by gentle vortexing.
Incubate tubes at 37°C for 0h 30m 0s.
Place the tubes on a magnet stand until the fluid is clear.
Transfer the supernatant containing the pA/G-MNase-bound digested chromatin fragments to fresh 1.5mLmicrocentrifuge tubes.
VIII. DNA extraction
Add 2µL 10% SDS to a final concentration of 0.1% and 5µL Proteinase K (10mg/mL) to a final concentration of 2.5mg/mL to each supernatant from step 62.
Gently vortex tubes at a low speed of approximately 1100rpm.
Incubate tubes at 50°C for 1h 0m 0s or at 37°C
Add 200µL PCI to tube.
Vortex tubes thoroughly at high speed until the liquid appears milky.
Optional: Transfer liquid to a phase-lock tube.
Centrifuge tubes in a table-top centrifuge at 16000x g at 4°C for 0h 5m 0s.
Carefully transfer the upper aqueous phase to a fresh 1.5mL microcentrifuge tube containing 200µL Chloroform:Isoamyl Alcohol 24:1 .
Vortex tubes thoroughly at high speed until the liquid appears milky.
Centrifuge tubes in a table-top centrifuge at 16000x g at 4°C for 0h 5m 0s.
Carefully transfer the upper aqueous phase to a fresh 1.5mL microcentrifuge
tube containing 2µL glycogen (diluted 1:10 to 2mg/mLfrom the20mg/mL
stock solution).
Add 20µL 3Molarity (M) NaOAc or 100µL 5Molarity (M) NH4OAc 4OAc
Add500µL 100% ethanol .
Place tubes for 0h 10m 0s in a dry ice/Ethanol mix or at -20°C.
Centrifuge tubes in a table-top centrifuge at 16000x g at 4°C for 0h 5m 0s.
Remove the liquid carefully with a pipette.
Add 1mL 70% ethanol .
Centrifuge tubes in a table-top centrifuge at 16000x g at 4°C for 0h 1m 0s.
Remove the liquid carefully with a pipette.
Air-dry the pellet or dry the pellet in a SpeedVac.
Dissolve the pellet in 30µL``1millimolar (mM) Tris-HCl , 0.1millimolar (mM) EDTA .
IX. Sample quality control
Size distribution and concentration of the CUT&RUN products can be assessed at this point, e.g. using a
Equipment
| Value | Label |
|---|---|
| Qubit | NAME |
| Flurometer | TYPE |
| Invitrogen | BRAND |
| Q33228 | SKU |
or
Equipment
| Value | Label |
|---|---|
| NanoDrop™ 3300 Fluorospectrometer | NAME |
| Fluorospectrometer | TYPE |
| NanoDrop™ 3300 | BRAND |
| nd-3300 | SKU |
or a Bioanalyzer or Tapestation. It is possible that the concentration of the recovered DNA is below the instrument’s detection limit. It is also to be expected that the extracted DNA includes some large DNA fragments that will mask the signal of the CUT&RUN products. In this case it may be useful to PCR-amplify the DNA and check the library on a Bioanalyzer or Tapestation.
X. Sequencing library preparation
Prepare the CUT&RUN products sequencing libraries according to your established work-
flow. Because of the very low background with CUT&RUN, typically 5 million paired-end
reads suffice for epitopes with a multitude of genomic binding sites, e.g. transcription
factors or nucleosome modifications.
XI. Peak calling
The sparse background signal in CUT&RUN samples compared to ChIP-seq samples
represents challenge for peak callers that employ statistical models relying on a high
sequencing depth and high recall to identify true positives and avoid false positives. In
contrast, peak calling for CUT&RUN data sets requires high specificity for true signal peaks.
To this end, the Henikoff lab developed the Sparse Enrichments analysis for CUT&RUN
(SEACR) peak caller that can be easily accessed using their web server at
Alternatively, the Orkin and Yuan labs have streamlined processing of CUT&RUN data using their CUT&RUNTools pipeline https://bitbucket.org/qzhudfci/cutruntools/
