Mar 19, 2020
Version 5
Bench top CUT&RUN with antibodies-online™ CUT&RUN Sets V.5
Version 1 is forked from Bench top CUT&Tag
- 1antibodies-online
- antibodies-onlineTech. support phone: +49 0241 95163153 email: support@antibodies-online.com
External link: https://campaigns.antibodies-online.com/cutandrun-sets/?utm_source=protocolsio&utm_medium=protocol&utm_content=cutrun
Protocol Citation: Antibodies Online Gmbh 2020. Bench top CUT&RUN with antibodies-online™ CUT&RUN Sets. protocols.io https://dx.doi.org/10.17504/protocols.io.bdwni7de
Manuscript citation:
The protocol is based on Skene; Henikoff JG; Henikoff S (2018): "Targeted in situ genome-wide profiling with high efficiency for low cell numbers"
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 19, 2020
Last Modified: March 19, 2020
Protocol Integer ID: 34478
Keywords: CUT&Tag, CUT&RUN, bench top protocol, epigenetics
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.
Guidelines
Frequently Asked Questions
Why is the DNA yield so low?
CUT&RUN is performed using low cell numbers and the background signal is considerable lower than e.g. for ChIP. Due to these two factors the amount of recovered DNA is often times too low to be reliably measured based on a fluorometric assay or by capillary electrophoresis. PCR amplification of small CUT&RUN products, i.e. less than 50 bp, can be problematic and is therefore not any option.
In order to assess the success of the CUT&RUN method each of the antibodies-online CUT&RUN product sets includes the CUT&RUN Positive Control antibody ABIN6923144•against the abundant H3K27me3 histone modification. DNA fragments prepared using this antibody can be measured by sensitive electrophoresis on a Bioanalyzer or Tapestation or fluorometrically on a Qubit or Nanodrop fluorometer. When using the CUT&RUN Positive Control antibody ABIN6923144•(or any other antibody specific for nucleosomal markers) a ladder corresponding to multiples of the 147 bp long nucleosomes should be visible by capillary electrophoresis.
How can I validate that my primary antibody is working for CUT&RUN?
For a CUT&RUN experiment the validation data could include e.g. a Tapestation or Bionalyzer plot showing the size distribution and qPCR data showing target enrichment.
As mentioned above, the DNA yield of an CUT&RUN experiment appears typically very low compared e.g. to ChIP-seq because of the lower initial sample size and the substantially lower DNA background. In particular for less abundant target protein the concentration is often times too low to be reliably measured using a fluorometric assay or by capillary
electrophoresis. PCR amplification of small CUT&RUN products, i.e. less than 50 bp, can be problematic and is therefore not an option.
Once a sequencing library has been generated and sequenced map sequencing reads and
verify the accumulation of reads at known binding sites.
Why do I need a negative control antibody? Why not just use a no-antibody control?
MNase is an endo- and exonuclease that will unspecifically bind and cleave unprotected DNA in hyper-accessible DNA, e.g. in regions surrounding regulatory elements. Free MNase will preferentially cut DNA within these hyper-accessible regions, thus potentially causing false positives and increased background signal in general.
In order to avoid this undesired effect of untethered MNase, the chromatin is randomly coated with the CUT&RUN Negative Control (ABIN6923140•) prior to the addition of pA/G-MNase is added to the samples. pA/G-MNase is then tethered via its Protein A or Protein G portion to the antbodies’ Fc fragment and background DNA fragmentation is dictated by the random antibody binding as opposed to the nuclease digestion of hyper-accessible DNA regions.
Can I replace the antibody negative control using a knock-out (or knock-down) of my protein?
Both controls are useful but address different aspects of the experiment and are therefore not interchangeable.
The CUT&RUN Negative Control (ABIN6923140) antibody is used to establish a reference background for peak calling. This is necessary because of the sparse background signal in CUT&RUN samples compared to ChIP-seq samples. The ko (or kd) control on the other hand gives an impression of unspecific binding of the antibody specific for the prote-
in of interest to other proteins. It is useful to avoid identification of false positive signals.
Do I need to use a secondary antibody? Other CUT&RUN protocols do not use a secondary.
Depending on the host species and isotype of the antibody and the Protein A and/or Protein G MNase fusion protein, a secondary antibody may be necessary for pA/G-MNase binding.
Protein A has good high affinity to all rabbit IgG antibodies but low affinity to rat, goat and sheep IgG isotype antibodies and certain mouse IgG antibody subclasses, in particular IgG1. Protein G on the other hand binds well to the Fc region of mouse, goat, sheep, and most rat IgG. Its affinity to rabbit IgG however is lower than that of Protein A. When using pAG-MNase introduced with the improved CUT&RUN protocol it is therefore generally not necessary to use a secondary antibody. Use of the pA-MNase of the original protocol however might require the use of a secondary antibody raised in rabbit to assure efficient binding of the fusion protein to the antibody.
Should I include heterologous spike-in DNA for quantitation?
Our protocol is largely based on the improved CUT&RUN protocol. Here, the authors show that accurate quantitation is possible using heterologous spike-in DNA or carry-over E. coli DNA from the pA/G-MNase purification.
Are there protocols to make this work for tissue, where I cannot necessarily isolate single cells?
The Henikoff lab that published the original CUT&RUN method in 2017 has since developed several variations. One of these modification is AutoCUT&RUN which outlines a workflow to automate CUT&RUN for high-throughput characterization of single cells but also patient samples and xenografts.
In short, the tissue is processed manually or enyzmatically into single, intact cell that are subsequently bound to ConA beads. The improved (and standard) CUT&RUN protocol that serve as basis for the protocol outlined in our CUT&RUN handbook can be adapted accordingly. Presently there is no work published documenting the use of intact tissue.
Is it possible to use the CUT&RUN product sets with plant tissue samples?
The CUT&RUN method can be applied without a problem to plant tissue samples (see e.g.PMID30719569). One essential step in addition to those lined out in the protocol is the generation of spheroblasts so that it becomes possible to permeabilize the plasma membrane for the application of the antibodies and the MNase fusion protein.
The positive control H3K27me3 antibody and the negative control guinea pig anti-rabbit IgG antibody as well as the ConA beads are suitable for use with plant samples. The antibodies that are included in some of the sets, such as the anti-DYKDDDDK antibodies or the secondary antibodies, can also be used with plant samples. Whether they are needed or not depends on your experiment.
Can CUT&RUN be adapted for RIP-seq?
It should be possible to adapt the CUT&RUN protocol for use with RNA as an alternative to RIP-seq. RNA in the cytoplasm will attract the degradation machinery if it is lacking the 5' cap and the 3' poly-A tail. Thus, it is advisable to use isolated nuclei as sample material. Consequently, no digitonin in the different buffers is needed since the nuclear envelope does not contain cholesterol. Isolated nuclei may be immobilized via glycoproteins on the nuclear envelope to the ConA beads like for a CUT&RUN experiment. An antibody against the protein of interest is added and subsequently the pA/G-MNase is tethered to the antibody, thus bringing the MNase into proximity of the RNA of interest. Similar to RIP-seq, isolated RNA is then translated into cDNA and can be sequenced and mapped.
Is it possible to use a cell free extract of a bacterial cell instead of immobilizing and permeabilizing the cell?
It should indeed be possible to use a bacterial extract as starting material instead of immobilized eukaryotic cells. There are a couple of things that should be considered:
One of the factors that makes CUT&RUN so much better than ChIP-seq in a eukaryotic system is that it is done in situ. The intact cells (or isolated nuclei) retain the large, undigested chromatin. Consequently, much fewer off-sites are present in the prepared DNA which is one of the reasons why CUT&RUN data sets have so much less background signal when compared to ChIP-seq data. This enrichment step will be missing when using a cell lysate as starting material. The CUT&RUN result should still be superior to the CHIP-seq result for the same starting material. However, it will be more "noisy" than CUT&RUN sequencing data from intact eukaryotic cells/nuclei.
The buffers described in the CUT&RUN protocol are designed to maintain the cells in the samples intact. The binding buffer contains some divalent cations that are necessary for the binding of the cells to the Concanavalin on the beads that are used for the immobilization. Subsequently, this buffer is changed for an antibody buffer that contains EDTA to chelate the divalent cations and prevent DNA cleavage prior to pA/G-MNase antibody binding. Since you are working with cell lysates as opposed to intact cells there is no need to include digitonin in any of the buffers. You don't need the binding buffer used to immobilize cells on the ConA beads. In order to control premature DNA cleavage by the MNase I would include some EDTA and/or EGTA in the lysis buffer.
Probably, you will have to titrate the amount of pA/G-MNase and try different digestion times to find the best balance between efficient cleavage of the intended sites and too much off-site cleavage.
The original CUT&RUN protocol foresees the use of heterologous spike-in DNA, e.g. from E. coli. In its latest iteration it is argued that there is no need to add this DNA since the pA/G-MNase preparation contains sufficient levels ofE. coli DNA to serve as a standard for quantitation. In a prokaryotic system, spike-in DNA from a eukaryotic organism (e.g.S. cerevisiae) is recommended. Also keep in in mind that theE. coli DNA carried over with the pA/G-MNase preparation may contain sequences that show homology to the bindings sites of your protein of interest.
Our product sets are optimized for use with eukaryotic cells. The positive control in all sets is a recombinant rabbit anti-H3K27me3 antibody. This control is not suitable for use with prokaryotic samples.
Instead of the proteinase K digestion can I denature the proteins in the CUT&RUN product complexes by heat?
Proteinase K digest followed by a phenol-chloroform extraction has historically been the go-to method to prepare high-molecular weight DNA, primarily from tissues. Subsequently to the diffusion of the CUT&RUN products out of the cells the DNA is present already in a relatively clean form, in particular when compared to a cell lysate like in ChIP-seq. Therefore, it is reasonable to assume that it is possible to forgo the Proteinase K treatment of the cells and instead proceed with the PCI extraction. As a reference, nucleic acid preparation using TRIzol does not include a Proteinase K digest. However, it has also been reported, that a proteinase K digest improves subsequent PCR amplification. Regarding heat inactivation instead of proteinase K digest in step, we recommend againstthis option. Your DNA of interest is at his point present in a complex consisting of the DNA, your antigen, the corresponding antibody, and the pA/G-MNase. If at this stage you boil your sample denature proteins you risk precipitation of your DNA with the protein compounds in this complex upon denaturation. This will also primarily affect the short CUT&RUN products, thus decreasing the signal to noise ratio in your library and potentially also reducing the library’s complexity.
What is preferable for DNA extractions prior to library preparation: phenol-chloroform extraction or affinity purification using a column?
In the original publication describing the CUT&Tag method the authors mention the use of AMPure XP beads for the purification of the DNA subsequently to tagmentation and Proteinase K digest. A potential issue is the carry-over of active Proteinase K, which can interfere with the downstream PCR amplification. Therefore, the authors recommend now the phenol-chloroform extraction to assure complete denaturation of Proteinase K.
Materials
MATERIALS
antibodies-online™ CUT&RUN Pro Complete Setantibodies-onlineCatalog #ABIN6923135
antibodies-online™ CUT&RUN Negative Controlantibodies-onlineCatalog #ABIN6923140
antibodies-online™ CUT&RUN Positive Controlantibodies-onlineCatalog #ABIN6923144
antibodies-online™ CUT&RUN Concanavalin A Beadsantibodies-onlineCatalog #ABIN6923139
antibodies-online™ CUT&RUN Secondaryantibodies-onlineCatalog #ABIN6923141
0.5 M EDTA pH 8.0antibodies-onlineCatalog # ABIN925554
Trypan Blue (0.4%)antibodies-onlineCatalog #ABIN413910
Proteinase Kantibodies-onlineCatalog #ABIN920948
CUT&RUN Pro Sec Setantibodies-onlineCatalog #ABIN6923137
Spike-in Chromatinantibodies-onlineCatalog #ABIN4889666
CUTANA™ pAG-MNase for ChIC/CUT&RUN Assaysantibodies-onlineCatalog #ABIN6950951
STEP MATERIALS
antibodies-online™ CUT&RUN Concanavalin A Beadsantibodies-onlineCatalog #ABIN6923139
antibodies-online™ CUT&RUN Positive Controlantibodies-onlineCatalog #ABIN6923144
antibodies-online™ CUT&RUN Negative Controlantibodies-onlineCatalog #ABIN6923140
antibodies-online™ CUT&RUN Secondaryantibodies-onlineCatalog #ABIN6923141
CUTANA™ pAG-MNase for ChIC/CUT&RUN Assaysantibodies-onlineCatalog #ABIN6950951
Reagents required
CUT&RUN Pro Sec Set (antibodies-online, cat no.ABIN6923137) with following components:
- Positive control Recombinant Rabbit anti-H3K27me3 Antibody (ABIN6923144)
- Negative control Polyclonal Guinea Pig anti-Rabbit IgG Antibody (ABIN6923140)
- Concanavalin A Beads (ABIN6923139)
- Secondary Rabbit anti-Mouse IgG (H&L) Antibody (ABIN6923141)
- Antibody to an epitope of interest
- Distilled, deionized or RNAse-free H2O
- 1 M Manganese Chloride (MnCl2)
- 1 M Calcium Chloride (CaCl2)
- 1 M Potassium Chloride
- 1 M HEPES pH 7.5 HEPES (NaOH)
- 5 M NaCl
- 0.5 M EDTA (ABIN925554)
- 0.2 M EGTA
- 2 M Spermidine
- Protease Inhibitor Cocktail
- 5% Digitonin (ABIN1304051)
- 20 mg/ml Glycogen
- Trypan Blue (ABIN413910)
- RNase A (DNase and protease free)
- 10% Sodium dodecyl sulfate (SDS)
- Proteinase K (ABIN920948)
- Phenol-chloroform-isoamyl alcohol (PCI)
- 7.5 M Ammonium Acetate (NH4OAc)
- 70 % Ethanol
- 100% Ethanol
- 1 mM Tris-HCl pH 8.0
Materials not provided with the package
CUTANA™ pAG-MNase for ChIC/CUT&RUN Assays ABIN6950951/ABIN6950952
Spike-in DNA 200 bp mean size (e.g, D. melanogaster, ABIN4889666)
Protocol materials
Proteinase Kantibodies-onlineCatalog #ABIN920948
Materials
antibodies-online™ CUT&RUN Concanavalin A Beadsantibodies-onlineCatalog #ABIN6923139
In Materials, Materials, Step 9
antibodies-online™ CUT&RUN Negative Controlantibodies-onlineCatalog #ABIN6923140
In Materials, Materials, Step 25
antibodies-online™ CUT&RUN Secondaryantibodies-onlineCatalog #ABIN6923141
In Materials, Materials, Step 35
antibodies-online™ CUT&RUN Positive Controlantibodies-onlineCatalog #ABIN6923144
In Materials, Materials, Step 25
Spike-in Chromatinantibodies-onlineCatalog #ABIN4889666
Materials
Trypan Blue (0.4%)antibodies-onlineCatalog #ABIN413910
Materials
0.5 M EDTA pH 8.0antibodies-onlineCatalog # ABIN925554
Materials
antibodies-online™ CUT&RUN Pro Complete Setantibodies-onlineCatalog #ABIN6923135
Materials
CUTANA™ pAG-MNase for ChIC/CUT&RUN Assaysantibodies-onlineCatalog #ABIN6950951
In Materials, Materials, Step 43
CUT&RUN Pro Sec Setantibodies-onlineCatalog #ABIN6923137
Materials
Safety warnings
Storage
Short term storage of the antibodies for up to two weeks at 4 °C. For long term storage for
up to one year at -20 °C, prepare 20 µL aliquots of the antibodies to avoid repeated free-
ze-thaw-cycles.
CUT&RUN Concanavalin A Beads must be stored at 4C. Do not freeze CUT&RUN
Concanavalin A Beads at -20 °C.
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.
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.
REAGENT SETUP (for 8 samples)
REAGENT SETUP (for 8 samples)
» Wash buffer (110 mL)
| Component | Volume | Final concentration | |
| ddH2O | 103 mL | - | |
| 1 M HEPES pH 7.5 | 2.2 mL | 20 mM | |
| 5 M NaCl | 3.3 mL | 150 mM | |
| 2 M Spermidine | 27.5 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.:
1.1 mL Protease Inhibitor Cocktail 100x
» Binding Buffer (30 mL)
| Component | Volume | Final concentration | |
| ddH2O | 29 mL | - | |
| 1 M HEPES pH 7.5 | 600 µL | 20 mM | |
| 1 M KCl | 300 µL | 10 mM | |
| 1 M CaCl2 | 30 µL | 1 mM | |
| 2.5 M MnCl2 | 12 µL | 1 mM |
• Store Binding Buffer for up to six months at 4 °C.
» Digitonin Wash Buffer (55 mL)
| Component | Volume | Final concentration | |
| 5 % Digitonin | 550 µL | 0.05 % | |
| Wash Buffer | 54.5 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 mL)
| Component | Volume | Final concentration | |
| 0.5 M EDTA | 4 µL | 2 mM | |
| 10 % BSA | 10 µL | 0.1 % | |
| Digitonin Wash Buffer | 986 µL | - |
• Store Antibody Buffer for up to one day at 4 °C until use.
» Low Salt Rinse Buffer (18 mL)
| Component | Volume | Final concentration | |
| ddH2O | 17.5 mL | - | |
| 1 M HEPES pH 7.5 | 360 µL | 20 mM | |
| 2 M Spermidine | 4,5 µL | 0.5 mM | |
| 5% Digitonin | 180 µL | 0.05 % |
• Store Low Salt Rinse Buffer for up to one week at 4 °C until use.
» Low Salt Incubation Buffer (2 mL)
| Component | Volume | Final concentration | |
| ddH2O | 1953 µL | - | |
| 1 M HEPES pH 7.5 | 7 µL | 3.5 mM | |
| 1 M CaCl2 | 20 µL | 10 mM | |
| 5% Digitonin | 20 µL | 0.05% |
• Store Low Salt Incubation Buffer for up to one week at 4 °C until use.
» Low Salt Stop Buffer (2 mL)
| Component | Volume | Final concentration | |
| ddH2O | 1700 mL | - | |
| 5 M NaCl | 68 µL | 170 mM | |
| 0.2 M EGTA | 200 µL | 20 mM | |
| Store Low Salt Stop Buffer at 4 °C until use. | |||
| Add fresh before use: | |||
| 5% Digitonin | 20 µL | 0.05% | |
| RNase A (10 mg/mL) | 10 µL | 50 µg/mL | |
| Glycogen (20 mg/mL) | 2.5 µL | 25 µg/mL | |
| Optional: | |||
| heterologous spike-in DNA | - | 100 pg/mL | |
I.Cell Harvest – at room temperature
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 solution00:03:00 at 600 x g at Room temperature . Remove the liquid carefully.
Gently resuspend cells in 1 mL Wash Buffer by pipetting and transfer cell solution to a 1.5 mL
microcentrifuge tube.
Centrifuge cell solution 00:03:00 at 600 x g at Room temperature and discard the supernatant.
Repeat steps 4-5 thrice for a total of four washes. go to step #4
Resuspend cell pellet for each sample in1 mL Wash Buffer by gently pipetting.
II. Concanavalin A beads preparation
II. Concanavalin A beads preparation
Prepare one 1.5 mL microcentrifuge tube for each sample.
Gently resuspend the
antibodies-online™ CUT&RUN Concanavalin A Beadsantibodies-onlineCatalog #ABIN6923139
Pipette 10 µL CUT&RUN Concanavalin A Beads slurry for each sample into the 1.5 mL 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.
Pipette1 mL 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 100 x 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 go to step #13 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. 10 µL per sample.
III. Cell immobilization – binding to Concanavalin A beads
III. Cell immobilization – binding to Concanavalin A beads
Carefully vortex the cell suspension from step go to step #7 and add 10 µL of the CUT&RUN Concanavalin A Beads in Binding Buffer prepared in section II to each sample.
Close tubes tightly and rotate for 00:05:00 to 00:10:00 at Room temperature .
IV. Cell permeabilization and primary antibody binding
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 1100 rpm ) and add 100 µL Antibody Buffer containing digitonin.
Gently vortex the microcentrifuge tubes until the beads are resuspended.
Add 1 µL primary antibody - against your antigen of interest, the
antibodies-online™ CUT&RUN Positive Controlantibodies-onlineCatalog #ABIN6923144
, and the
antibodies-online™ CUT&RUN Negative Controlantibodies-onlineCatalog #ABIN6923140
- corresponding to a 1:100 dilution (or a volume corresponding to the manufacturer’s recom- mended dilution for immunofluorescence).
Rotate the microcentrifuge tubes for 00:05:00 to 00:10:00 at Room temperature or 02:00:00 toOvernight 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 1 mL Digitonin Wash Buffer and mix by inversion. If clumping occurs, gently remove the clumps with a 1 mL pipette tip.
Repeat steps 27-29 go to step #27 once for a total of two washes.
Note
If no secondary antibody is used proceed directly to section VI. pA/G-MNase binding (Step 41).
V. Secondary antibody binding (optional)
V. Secondary antibody binding (optional)
Safety information
The
antibodies-online™ CUT&RUN Secondaryantibodies-onlineCatalog #ABIN6923141
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.
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 1100 rpm ) and add 100 µL Digitonin Wash Buffer per sample along the side of the tube.
Tap to remove the remaining beads from the tube side.
Add 1 µL
antibodies-online™ CUT&RUN Secondaryantibodies-onlineCatalog #ABIN6923141
corresponding to a 1:100 dilution (or a volume corresponding to the manufacturer’s recommended dilution for immunofluorescence).
Rotate the microcentrifuge tubes for 01:00:00 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 1 mL Digitonin Wash Buffer and mix by inversion. If clumping occurs, gently remove the clumps with a 1 mL pipette tip.
Repeat steps 37-39 go to step #33 once for a total of two washes.
VI. Protein A-MNase or Protein AG-MNase Binding
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 1100 rpm ) and add 50 µL Digitonin Wash Buffer per sample along the side of the tube. Add 2.5 µL
CUTANA™ pAG-MNase for ChIC/CUT&RUN Assaysantibodies-onlineCatalog #ABIN6950951
Alternatively:
Vortex the sample at low speed (approximately 1100 rpm ) and add 150 µL Digitonin Wash Buffer containing 700 ng/mL of your own pA/G-MNase preparation per sample along the side of the tube.
Rotate the microcentrifuge tubes for 01:00:00 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 1 mL Digitonin Wash Buffer and mix by inversion. If clumping occurs, gently remove the clumps with a 1 ml pipette tip.
Repeat steps 45-47 go to step #45 once for a total of two washes.
VII. MNase digestion and release of pA/G bound chromatin fragments
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 100 x g ).
Place the tubes on a magnet stand until the fluid is clear. Remove the liquid carefully.
Resuspend with 1 mL 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 100 x g ).
Place the tubes on a magnet stand until the fluid is clear. Remove the liquid carefully.
Repeat steps 51-53 go to step #51 once for a total of two washes.
Place each tube at a low angle on the vortex mixer set to a low speed (approx. 1100 rpm ) and add 200 µL 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 00:05:00 ).
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 200 µL Low Salt Stop Solution and mix by gentle vortexing.
Incubate tubes at 37 °C for 00:30:00 .
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 1.5 mL microcentrifuge tubes.
VIII. DNA extraction
VIII. DNA extraction
Add 2 µL 10% SDS to a final concentration of 0.1% and 5 µL Proteinase K (10 mg/mL ) to a final concentration of 2.5 mg/mL to each supernatant from step 62.
Gently vortex tubes at a low speed of approximately 1100 rpm .
Incubate tubes at 50 °C for 01:00:00 or at 37 °C Overnight
Add 200 µL 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 16000 x g at 4 °C for 00:05:00 .
Carefully transfer the upper aqueous phase to a fresh 1.5 mL microcentrifuge tube containing 200 µL Chloroform:Isoamyl Alcohol 24:1.
Vortex tubes thoroughly at high speed until the liquid appears milky.
Centrifuge tubes in a table-top centrifuge at 16000 x g at 4 °C for 00:05:00 .
Carefully transfer the upper aqueous phase to a fresh 1.5 mL microcentrifuge
tube containing 2 µL glycogen (diluted 1:10 to 2 mg/mL from the20 mg/mL
stock solution).
Add 20 µL 3 Molarity (M) NaOAc or 100 µL 5 Molarity (M) NH4OAc
Add500 µL 100% ethanol.
Place tubes for 00:10:00 in a dry ice/Ethanol mix or Overnight at -20 °C .
Centrifuge tubes in a table-top centrifuge at 16000 x g at 4 °C for 00:05:00 .
Remove the liquid carefully with a pipette.
Add 1 mL 70% ethanol.
Centrifuge tubes in a table-top centrifuge at 16000 x g at 4 °C for 00:01:00 .
Remove the liquid carefully with a pipette.
Air-dry the pellet or dry the pellet in a SpeedVac.
Dissolve the pellet in 30 µL 1 millimolar (mM) Tris-HCl, 0.1 millimolar (mM) EDTA.
IX. Sample quality control
IX. Sample quality control
Size distribution and concentration of the CUT&RUN products can be assessed at this point, e.g. using a
Equipment
Qubit
NAME
Flurometer
TYPE
Invitrogen
BRAND
Q33228
SKU
LINK
or
Equipment
NanoDrop™ 3300 Fluorospectrometer
NAME
Fluorospectrometer
TYPE
NanoDrop™ 3300
BRAND
nd-3300
SKU
LINK
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
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
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/