TITLE: Bench top CUT&RUN with antibodies-online™ CUT&RUN Sets
AUTHORS: Antibodies Online Gmbh

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">CUT&RUN (Cleavage Under Targets and Release Using Nuclease) offers a novel approach </div><div class = "text-block">to pursue epigenetics. The method is designed to map genome wide transcription factor </div><div class = "text-block">binding sites, chromatin-associated complexes, and histone variants and post-translational </div><div class = "text-block">modifications.</div><div class = "text-block">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.</div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/protocols-files/public/0a4fe9b7a11f73c8ba5166c11a7060d8420e56909f0bef279aef656fabd5f2ab/btss9xr6.png" /></div><div class = "text-block">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).</div><div class = "text-block">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. </div></div>

[STEPS]
?. [ REAGENT SETUP (for 8 samples)]
» Wash buffer (110 mL)  ABC1ComponentVolumeFinal concentration2ddH2O103 mL-31 M HEPES pH 7.5 2.2 mL20 mM45 M NaCl 3.3 mL150 mM52 M Spermidine 27.5 µL 0.5 mM» Binding Buffer (30 mL)  ABC1ComponentVolumeFinal concentration2ddH2O29 mL-31 M HEPES pH 7.5600 µL20 mM41 M KCl300 µL10 mM51 M CaCl230 µL1 mM62.5 M MnCl212 µL1 mM» Digitonin Wash Buffer (55 mL)  ABC1ComponentVolumeFinal concentration25 % Digitonin550 µL0.05 %3Wash Buffer54.5 mL-» Antibody Buffer (1 mL)  ABC1ComponentVolumeFinal concentration20.5 M EDTA4 µL2 mM310 % BSA10 µL0.1 %4Digitonin Wash Buffer986 µL- » Low Salt Rinse Buffer (18 mL)  ABC1ComponentVolumeFinal concentration2ddH2O17.5 mL-31 M HEPES pH 7.5360 µL20 mM42 M Spermidine4,5 µL0.5 mM55% Digitonin180 µL0.05 %» Low Salt Incubation Buffer (2 mL)  ABC1ComponentVolumeFinal concentration2ddH2O 1953 µL-31 M HEPES pH 7.57 µL3.5 mM41 M CaCl2 20 µL10 mM55% Digitonin20 µL0.05%» Low Salt Stop Buffer (2 mL)  ABC1ComponentVolumeFinal concentration2ddH2O 1700 mL-35 M NaCl 68 µL170 mM40.2 M EGTA200 µL20 mM5Store Low Salt Stop Buffer at 4 °C until use.Store Low Salt Stop Buffer at 4 °C until use.Store Low Salt Stop Buffer at 4 °C until use.6Add fresh before use:75% Digitonin20 µL0.05%8RNase A (10 mg/mL)10 µL50 µg/mL9Glycogen (20 mg/mL)2.5 µL25 µg/mL10Optional:11heterologous spike-in DNA-100 pg/mL
ABC1ComponentVolumeFinal concentration2ddH2O103 mL-31 M HEPES pH 7.5 2.2 mL20 mM45 M NaCl 3.3 mL150 mM52 M Spermidine 27.5 µL 0.5 mM
ABC1ComponentVolumeFinal concentration2ddH2O29 mL-31 M HEPES pH 7.5600 µL20 mM41 M KCl300 µL10 mM51 M CaCl230 µL1 mM62.5 M MnCl212 µL1 mM
ABC1ComponentVolumeFinal concentration25 % Digitonin550 µL0.05 %3Wash Buffer54.5 mL-
ABC1ComponentVolumeFinal concentration20.5 M EDTA4 µL2 mM310 % BSA10 µL0.1 %4Digitonin Wash Buffer986 µL-
ABC1ComponentVolumeFinal concentration2ddH2O17.5 mL-31 M HEPES pH 7.5360 µL20 mM42 M Spermidine4,5 µL0.5 mM55% Digitonin180 µL0.05 %
ABC1ComponentVolumeFinal concentration2ddH2O 1953 µL-31 M HEPES pH 7.57 µL3.5 mM41 M CaCl2 20 µL10 mM55% Digitonin20 µL0.05%
ABC1ComponentVolumeFinal concentration2ddH2O 1700 mL-35 M NaCl 68 µL170 mM40.2 M EGTA200 µL20 mM5Store Low Salt Stop Buffer at 4 °C until use.Store Low Salt Stop Buffer at 4 °C until use.Store Low Salt Stop Buffer at 4 °C until use.6Add fresh before use:75% Digitonin20 µL0.05%8RNase A (10 mg/mL)10 µL50 µg/mL9Glycogen (20 mg/mL)2.5 µL25 µg/mL10Optional:11heterologous spike-in DNA-100 pg/mL
?. [I.Cell Harvest – at room temperature]
Harvest 10,000 to 500,000 cells for each sample at . Keep cells for each sample in separate tubes.
0 Room temperature
?. [I.Cell Harvest – at room temperature]
Centrifuge cell solution at  at . Remove the liquid carefully.
Centrifuge: 600 34
0 Room temperature
?. [I.Cell Harvest – at room temperature]
Gently resuspend cells in  Wash Buffer by pipetting and transfer cell solution to a microcentrifuge tube.
1 ml
1.5 ml
?. [I.Cell Harvest – at room temperature]
Centrifuge cell solution  at  at  and discard the supernatant.
Centrifuge: 600 34
0 Room temperature
?. [I.Cell Harvest – at room temperature]
Repeat steps 4-5 thrice for a total of four washes.
?. [I.Cell Harvest – at room temperature]
Resuspend cell pellet for each sample in Wash Buffer by gently pipetting.
1 ml
?. [II. Concanavalin A beads preparation ]
Prepare one  microcentrifuge tube for each sample.
1.5 ml
?. [II. Concanavalin A beads preparation ]
Gently resuspend the
?. [II. Concanavalin A beads preparation ]
Pipette  CUT&RUN Concanavalin A Beads slurry for each sample into the  microcentrifuge tubes.
10 µl
1.5 ml
?. [II. Concanavalin A beads preparation ]
Place the tubes on a magnet stand until the fluid is clear. Remove the liquid carefully.
?. [II. Concanavalin A beads preparation ]
Remove the microcentrifuge tube from the magnet stand.
?. [II. Concanavalin A beads preparation ]
Pipette Binding Buffer into each tube and resuspend CUT&RUN Concanavalin A Beads by gentle pipetting.
1 ml
?. [II. Concanavalin A beads preparation ]
Spin down the liquid from the lid with a quick pulse in a table-top centrifuge (max ).
Centrifuge: 100 34
?. [II. Concanavalin A beads preparation ]
Place the tubes on a magnet stand until the fluid is clear. Remove the liquid carefully.
?. [II. Concanavalin A beads preparation ]
Remove the microcentrifuge tube from the magnet stand.
?. [II. Concanavalin A beads preparation ]
Repeat steps 13-16  twice for a total of three washes.
?. [II. Concanavalin A beads preparation ]
Gently resuspend the CUT&RUN Concanavalin A Beads in a volume of Binding Buffer corresponding to the original volume of bead slurry, i.e.  per sample.
10 µl
?. [III. Cell immobilization – binding to Concanavalin A beads]
Carefully vortex the cell suspension from step  and add  of the CUT&RUN Concanavalin A Beads in Binding Buffer prepared in section II to each sample.
10 µl
?. [III. Cell immobilization – binding to Concanavalin A beads]
Close tubes tightly and rotate for  to  at .
0 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.
?. [IV. Cell permeabilization and primary antibody binding]
Remove the microcentrifuge tubes from the magnet stand.
?. [IV. Cell permeabilization and primary antibody binding]
Place each tube at a low angle on the vortex mixer set to a low speed (approximately ) and add  Antibody Buffer containing digitonin.
Centrifuge: 1100 33
100 µl
?. [IV. Cell permeabilization and primary antibody binding]
Gently vortex the microcentrifuge tubes until the beads are resuspended.
?. [IV. Cell permeabilization and primary antibody binding]
Add  primary antibody - against your antigen of interest, the  , and the - corresponding to a 1:100 dilution (or a volume corresponding to the manufacturer’s recom- mended dilution for immunofluorescence).
1 µl
?. [IV. Cell permeabilization and primary antibody binding]
Rotate the microcentrifuge tubes for  to  at  or  to at .
4 Room temperature
4 °C
?. [IV. Cell permeabilization and primary antibody binding]
Spin down the liquid and place the tubes on a magnet stand until the fluid is clear. Remove the liquid carefully.
?. [IV. Cell permeabilization and primary antibody binding]
Remove the microcentrifuge tubes from the magnet stand.
?. [IV. Cell permeabilization and primary antibody binding]
Resuspend with  Digitonin Wash Buffer and mix by inversion. If clumping occurs, gently remove the clumps with a  pipette tip.
1 ml
1 ml
?. [IV. Cell permeabilization and primary antibody binding]
Repeat steps 27-29   once for a total of  two washes.
If no secondary antibody is used proceed directly to section VI. pA/G-MNase binding (Step 41).
?. [V. Secondary antibody binding (optional)]
Place the tubes on a magnet stand until the fluid is clear. Remove the liquid carefully.
The  is raised in rabbit against mouse IgG (H&L). In case a primary antibody from a different host species or isotype is used a different secondary must be selected accordingly.
?. [V. Secondary antibody binding (optional)]
Remove the microcentrifuge tubes from the magnet stand.
?. [V. Secondary antibody binding (optional)]
Vortex the sample at low speed (approximately ) and add  Digitonin Wash Buffer per sample along the side of the tube.
Centrifuge: 1100 33
100 µl
?. [V. Secondary antibody binding (optional)]
Tap to remove the remaining beads from the tube side.
?. [V. Secondary antibody binding (optional)]
Add corresponding to a 1:100 dilution (or a volume corresponding to the manufacturer’s recommended dilution for immunofluorescence).
1 µl
?. [V. Secondary antibody binding (optional)]
Rotate the microcentrifuge tubes for  at .
4 °C
?. [V. Secondary antibody binding (optional)]
Spin down the liquid and place the tubes on a magnet stand until the fluid is clear. Remove the liquid carefully.
?. [V. Secondary antibody binding (optional)]
Remove the microcentrifuge tubes from the magnet stand.
?. [V. Secondary antibody binding (optional)]
Resuspend with  Digitonin Wash Buffer and mix by inversion. If clumping occurs, gently remove the clumps with a 1 mL pipette tip.
1 ml
?. [V. Secondary antibody binding (optional)]
Repeat steps 37-39   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.
?. [VI. Protein A-MNase or Protein AG-MNase Binding]
Remove the microcentrifuge tubes from the magnetic stand.
?. [VI. Protein A-MNase or Protein AG-MNase Binding]
Vortex the sample at low speed (approximately ) and add  Digitonin Wash Buffer  per sample along the side of the tube. Add Alternatively:Vortex the sample at low speed (approximately ) and add  Digitonin Wash Buffer  containing   of your own pA/G-MNase preparation per sample along the side of the tube.
Centrifuge: 1100 33
50 µl
2.5 µl
Centrifuge: 1100 33
150 µl
700
?. [VI. Protein A-MNase or Protein AG-MNase Binding]
Rotate the microcentrifuge tubes for  at .
4 °C
?. [VI. Protein A-MNase or Protein AG-MNase Binding]
Spin down the liquid and place the tubes on a magnet stand until the fluid is clear. Remove the liquid carefully.
?. [VI. Protein A-MNase or Protein AG-MNase Binding]
Remove the microcentrifuge tubes from the magnetic stand.
?. [VI. Protein A-MNase or Protein AG-MNase Binding]
Resuspend with  Digitonin Wash Buffer and mix by inversion. If clumping occurs, gently remove the clumps with a 1 ml pipette tip.
1 ml
?. [VI. Protein A-MNase or Protein AG-MNase Binding]
Repeat steps 45-47   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 ).
Centrifuge: 100 34
?. [VII. MNase digestion and release of pA/G bound chromatin fragments]
Place the tubes on a magnet stand until the fluid is clear. Remove the liquid carefully.
?. [VII. MNase digestion and release of pA/G bound chromatin fragments]
Resuspend with   Low Salt Rinse Buffer and mix by inversion. If clumping occurs, gently remove the clumps with a 1 mL pipette tip.
1 ml
?. [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 ).
Centrifuge: 100 34
?. [VII. MNase digestion and release of pA/G bound chromatin fragments]
Place the tubes on a magnet stand until the fluid is clear. Remove the liquid carefully.
?. [VII. MNase digestion and release of pA/G bound chromatin fragments]
Repeat steps 51-53   once for a total of two washes.
?. [VII. MNase digestion and release of pA/G bound chromatin fragments]
Place each tube at a low angle on the vortex mixer set to a low speed (approx.  ) and add  ice cold Low Salt Incubation Buffer per sample along the side of the tube.
1100
200 µl
?. [VII. MNase digestion and release of pA/G bound chromatin fragments]
Incubate tubes at  for the desired time (default is ).
0 °C
?. [VII. MNase digestion and release of pA/G bound chromatin fragments]
Place the tubes on a cold magnet stand until the fluid is clear. Remove the liquid carefully.
?. [VII. MNase digestion and release of pA/G bound chromatin fragments]
Remove the microcentrifuge tubes from the magnet stand.
?. [VII. MNase digestion and release of pA/G bound chromatin fragments]
Resuspend with   Low Salt Stop Solution and mix by gentle vortexing.
200 µl
?. [VII. MNase digestion and release of pA/G bound chromatin fragments]
Incubate tubes at  for .
37 °C
?. [VII. MNase digestion and release of pA/G bound chromatin fragments]
Place the tubes on a magnet stand until the fluid is clear.
?. [VII. MNase digestion and release of pA/G bound chromatin fragments]
Transfer the supernatant containing the pA/G-MNase-bound digested chromatin fragments to fresh microcentrifuge tubes.
1.5 ml
?. [VIII. DNA extraction]
Add  10% SDS to a final concentration of 0.1% and  Proteinase K () to a final concentration of  to each supernatant from step 62.
2 µl
5 µl
?. [VIII. DNA extraction]
Gently vortex tubes at a low speed of approximately .
Centrifuge: 1100 33
?. [VIII. DNA extraction]
Incubate tubes at  for  or at
50 °C
37 °C
?. [VIII. DNA extraction]
Add  PCI to tube.
200 µl
?. [VIII. DNA extraction]
Vortex tubes thoroughly at high speed until the liquid appears milky.
?. [VIII. DNA extraction]
Optional: Transfer liquid to a phase-lock tube.
?. [VIII. DNA extraction]
Centrifuge tubes in a table-top centrifuge at  at  for .
Centrifuge: 16000 34
4 °C
?. [VIII. DNA extraction]
Carefully transfer the upper aqueous phase to a fresh  microcentrifuge tube containing  Chloroform:Isoamyl Alcohol 24:1.
1.5 ml
200 µl
?. [VIII. DNA extraction]
Vortex tubes thoroughly at high speed until the liquid appears milky.
?. [VIII. DNA extraction]
Centrifuge tubes in a table-top centrifuge at  at  for .
Centrifuge: 16000 34
4 °C
?. [VIII. DNA extraction]
Carefully transfer the upper aqueous phase to a fresh   microcentrifuge tube containing  glycogen (diluted 1:10 to  from thestock solution).
1.5 ml
2 µl
2
20
?. [VIII. DNA extraction]
Add  NaOAc or  NH4OAc
20 µl
100 µl
?. [VIII. DNA extraction]
Add 100% ethanol.
500 µl
?. [VIII. DNA extraction]
Place tubes for  in a dry ice/Ethanol mix or  at .
-20 °C
?. [VIII. DNA extraction]
Centrifuge tubes in a table-top centrifuge at  at  for .
Centrifuge: 16000 34
4 °C
?. [VIII. DNA extraction]
Remove the liquid carefully with a pipette.
?. [VIII. DNA extraction]
Add  70% ethanol.
1 ml
?. [VIII. DNA extraction]
Centrifuge tubes in a table-top centrifuge at  at  for .
Centrifuge: 16000 34
4 °C
?. [VIII. DNA extraction]
Remove the liquid carefully with a pipette.
?. [VIII. DNA extraction]
Air-dry the pellet or dry the pellet in a SpeedVac.
?. [VIII. DNA extraction]
Dissolve the pellet in  Tris-HCl,  EDTA.
30 µl
?. [IX. Sample quality control]
Size distribution and concentration of the CUT&RUN products can be assessed at this point, e.g. using a  or  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  https://seacr.fredhutch.org/.Alternatively, the Orkin and Yuan labs have streamlined processing of CUT&RUN data using their CUT&RUNTools pipeline https://bitbucket.org/qzhudfci/cutruntools/