Mar 19, 2025
CUT&RUN protocol for activated primary B cells
- Stormy E. Ruiz1
- 1Laboratory of Molecular Biology and Immunology, National Institute on Aging, National Institutes of Health
Protocol Citation: Stormy E. Ruiz 2025. CUT&RUN protocol for activated primary B cells. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl491y2go5/v1
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: August 12, 2024
Last Modified: March 19, 2025
Protocol Integer ID: 105841
Keywords: CUT & RUN, Cleavage Under Targets Release Using Nuclease, B cells, Primary B cells
Funders Acknowledgements:
Intramural Research Program at the National Institutes of Health, National Institute on Aging
Grant ID: AG000714
Abstract
CUT&RUN is a useful tool for the study of interactions between proteins and chromatin in limited samples. However, this technique was originally designed using cell lines and for targeting histones. Activated primary B cells are subject to degradation using the original methodology, so the protocol here was optimized for use on these cells. Further, this method was adapted and validated for non-histone proteins, such as the RNA polymerase II complex. This protocol is suitable for use on subsets of primary B cells and to target non-histone protein targets and post-translational modifications.
Guidelines
- If you are stimulating cells fresh for CUT&RUN or want to prepare stimulated B cells for a later date, proceed to section titled B cell isolation.
- If you will be using previously frozen samples, proceed to section titled Thawing frozen samples.
Materials
Buffers:
Make all buffers fresh on day 1 of the CUT&RUN experiment and keep all at 4°C or on ice at all times – especially once spermidine is added.
Note
Prepare aliquots of 1 M spermidine on ice and freeze. Thaw fresh aliquots on ice each time it is needed and discard remainer of tube after thaw.
Pre-Nuclear Isolation Buffer:
- 20 mM HEPES, pH 7.9
- 10 mM KCl
- 1 mM MnCl2
- 0.1% Triton X-100
- 20% glycerol
Filter sterilize. Can be stored for up to 6 months at 4°C
Nuclear Isolation Buffer:
- Pre-Nuclear Isolation Buffer
- cOmplete EDTA-free Protease inhibitor
- PhosSTOP phosphatase inhibitor (if needed)
- 1 mM Spermidine
Pre-XLS Wash Buffer:
- Pre-Wash Buffer (20 mM HEPES-KOH pH 7.9, 150 mM NaCl) (in EpiCypher CUTANA CUT&RUN/ChIC kit)
- 1% Triton X-100
- 0.05% SDS
- Filter sterilize
XLS-Wash Buffer:
- Pre-XLS Wash Buffer
- cOmplete EDTA-free Protease inhibitor
- PhosSTOP phosphatase inhibitor (if needed)
- 1 mM Spermidine
XLS-Antibody Binding Buffer:
- XLS-Wash Buffer
- 2 mM EDTA
Materials and Equipment:
| A | B | C | |
| REAGENT or RESOURCE | SOURCE | IDENTIFIER | |
| Antibodies | |||
| H3K4me3 Antibody, SNAP-Certified for CUT&RUN and CUT&Tag | EpiCypher | Cat#13-0060 | |
| CUTANA IgG Negative Control Antibody for CUT&RUN and CUT&Tag | EpiCypher | Cat#13-0042 | |
| Anti-CD40 (mouse) mAb clone FGK45 | Enzo Life Sciences | Cat#ALX-805-046PF-C500; RRID: AB_10997445 | |
| Chemicals, peptides, and recombinant proteins | |||
| RPMI Medium 1640 | Gibco | Cat#11875-101 | |
| Fetal Bovine Serum, Heat Inactivated | Gibco | Cat#10-082-147 | |
| GlutaMAX Supplement | Gibco | Cat#35050061 | |
| Penicillin-Streptomycin (5,000 U/mL) | Gibco | Cat#15070063 | |
| Betamercaptoethanol | Sigma Life Science | Cat#M3148-25ML, CAS: 60-24-2 | |
| Lipopolysaccharide | Millipore Sigma | Cat#L2630-10MG | |
| Recombinant mouse IL-4 | R&D Systems | Cat#404-ML/CF | |
| ACK Lysing Buffer | Quality Biological | Cat#118-156-101 | |
| Dimethylsulfoxide (DMSO) | Invitrogen | Cat#D12345; CAS: 67-68-5 | |
| Dulbecco’s PBS (DPBS) | Gibco | Cat#14190-144 | |
| Sodium Dodecyl Sulfate (SDS), 20% w/v | Quality Biological | Cat#351-066-721; CAS: 151-21-3 | |
| Triton X-100 | Fisher Scientific | Cat#BP151-100, CAS: 9002-93-1 | |
| cOmplete protease inhibitor, EDTA-free | Roche | Cat#11873580001 | |
| HyPure Molecular Biology Grade Water | Cytiva | Cat#SH30538.03; CAS: 7732-18-5 | |
| Manganese Chloride | Fisher Scientific | Cat#M87-100; CAS: 13446-34-9 | |
| Proteinase K | Ambion | Cat#AM2548 | |
| Spermidine | Acros Organics | Cat#132740010; CAS: 124-20-9 | |
| HEPES | Sigma Life Science | Cat#H3375-250G; CAS: 7365-45-9 | |
| Glycerol, 50% v/v | Ricca Chemical | Cat#3290-32; CAS: 56-81-5 | |
| Formaldehyde, 37% by weight | Fisher Scientific | Cat#BP531-25; CAS: 50-00-0 | |
| Glycine | Fisher Scientific | Cat#BP381-1; CAS: 56-40-6 | |
| Trypan Blue | Invitrogen | Cat#T10282 | |
| Critical commercial assays | |||
| CUTANA ChIC/CUT&RUN Kit v1-4 | EpiCypher | Cat#14-1048 | |
| CUTANA CUT&RUN Library Prep Kit Primer Set 1 and Set 2 | EpiCypher | Cat#14-1001; Cat#14-1002 | |
| Dead Cell Removal Kit | Miltenyi Biotec | Cat#130-090-101 | |
| CD23 MicroBeads, mouse | Miltenyi Biotec | Cat#130-098-784 | |
| High Sensitivity DNA Reagents Kit for Bioanalyzer 2100 | Agilent Technologies | Cat#5067-4626 | |
| Qubit 1x dsDNA HS Assay Kit | Invitrogen | Cat#Q33231 | |
| NovaSeq 6000 SP Reagent Kit v1.5 (200 cycles) | Illumina | Cat#20040719 | |
| MiniSeq Mid Output Kit (300-cycles) | Illumina | Cat#FC-420-1004 | |
| SPRIselect | Beckman Coulter | Cat#B23318 | |
| Software and algorithms | |||
| BCL convert | Illumina | https://support.illumina.com/sequencing/sequencing_software/bcl-convert.html | |
| TrimGalore | DOI 10.5281/zenodo.5127898 | https://github.com/FelixKrueger/TrimGalore | |
| bowtie2 | Langmead and Salzberg | http://bowtie-bio.sourceforge.net/bowtie2/index.shtml | |
| SAMtools | Li, et al. | http://samtools.sourceforge.net/ | |
| MACS | Zhang, et.al. | https://macs3-project.github.io/MACS/ | |
| IDR | Li, et al. | https://github.com/nboley/idr | |
| Integrated Genomics Viewer (IGV) | Robinson, et. al. | https://igv.org/ | |
| Other | |||
| Agilent Bioanalyzer 2100 | Agilent Technologies | https://www.agilent.com/en/product/automated-electrophoresis/bioanalyzer-systems/bioanalyzer-instrument/2100-bioanalyzer-instrument-228250 | |
| Qubit 4 fluorometer | Invitrogen | Cat#Q33238 | |
| MiniSeq | Illumina | Cat#SY-420-1001 | |
| NovaSeq 6000 | Illumina | https://www.illumina.com/systems/sequencing-platforms/novaseq.html | |
| Veriti Thermal Cycler, 96-Well | Applied Biosystems | Cat#4375305 | |
| Strip-tube shaking block | Custom made in-house | N/A | |
| Mr. Frosty freezing container | ThermoFisher Scientific | Cat#5100-0001 | |
| Cell Strainer 70 µm Nylon | Falcon | Cat#352350 | |
| LS Columns | Miltenyi Biotec | Cat#130-042-401 | |
| MidiMACS Separator | Miltenyi Biotec | Cat#130-042-302 | |
| EpiCypher 8-strip PCR tubes | Epicypher | Cat#10-0009 | |
| 10X magnetic separator | 10x Genomics | Cat#120250 |
H3K4me3 Antibody, SNAP-Certified™ for CUT&RUN and CUT&TagEpiCypherCatalog #13-0060
CUTANA™ IgG Negative Control Antibody for CUT&RUN and CUT&TagEpiCypherCatalog #13-0042
CD40 (mouse) monoclonal antibody (FGK45)Enzo Life SciencesCatalog #ALX-805-046PF-C500
RPMI 1640 MediumThermo FisherCatalog #11875101
Heat-Inactivated FBS Gibco - Thermo FischerCatalog #10-082-147
GlutaMAX™ SupplementGibco - Thermo FisherCatalog #35050061
Penicillin-Streptomycin (5,000 U/mL)Thermo FisherCatalog #15070063
b-mercaptoethanolMerck MilliporeSigma (Sigma-Aldrich)Catalog #M3148-25ML
LipopolysaccharidesMerck MilliporeSigma (Sigma-Aldrich)Catalog #L2630-10MG
Recombinant Mouse IL-4 ProteinR&D SystemsCatalog #404-ML
ACK Lysing BufferQuality BiologicalCatalog #118-156-101
DMSO, AnhydrousThermo FisherCatalog #D12345
Dulbeccos Phosphate Buffered Solution [DPBS]Gibco - Thermo FisherCatalog #14190-144
SDS (20%)Quality BiologicalCatalog #351-066-721
Triton X-100Fisher ScientificCatalog #BP151-100
cOmplete™ EDTA-free Protease Inhibitor CocktailRocheCatalog #11873580001
Molecular Biology Grade WaterCytivaCatalog #SH30538.03
Manganese ChlorideFisher ScientificCatalog #M87-100
Proteinase KThermo Fisher ScientificCatalog #AM2548
Spermidine, 99%Acros OrganicsCatalog #132740010
HEPESMerck MilliporeSigma (Sigma-Aldrich)Catalog #H3375
Glycerin (Glycerol), 50% (v/v)Ricca Chemical CompanyCatalog #3290-32
Formaldehyde (37% by Weight/Molecular Biology), Fisher BioReagentsFisher ScientificCatalog #BP531-25
Glycine (White Crystals or Crystalline Powder)Fisher ScientificCatalog #BP381-1
Trypan BlueInvitrogen - Thermo FisherCatalog #T10282
Cutana ChIC/CUT&RUN KitEpiCypherCatalog #14-1048
CUTANA CUT&RUN Library Prep Kit Primer Set 1 and Set 2EpiCypherCatalog #14-1001 ; 14-1002
Dead Cell Removal KitMiltenyi BiotecCatalog #130-090-101
CD23 MicroBeads, mouseMiltenyi BiotecCatalog #130-098-784
Bioanalyzer High Sensitivity DNA KitAgilent TechnologiesCatalog #5067-4626
Qubit™ 1X dsDNA HS Assay KitInvitrogen - Thermo FisherCatalog #Q33231
NovaSeq 6000 SP Reagent Kit v1.5 (200 cycles)Illumina, Inc.Catalog #20040719
MiniSeq Mid Output Kit (300-cycles)Illumina, Inc.Catalog #FC-420-1004
SPRIselectBeckman CoulterCatalog #B23318
Qubit 4 FluorometerThermo Fisher ScientificCatalog #Q33238
Illumina MiniSeq High Output Reagent Kit Illumina, Inc.Catalog #FC-420-1001
Veriti™ 96-Well Fast Thermal CyclerThermo FisherCatalog #4375305
Mr. Frosty™ Freezing ContainerThermo Fisher ScientificCatalog #5100-0001
Cell strainer 70um filterFalconCatalog #352350
LS ColumnsMiltenyi BiotecCatalog #130-042-401
MidiMACS SeparatorMiltenyi BiotecCatalog #130-042-302
CUTANA™ CUT&RUN 8-strip 0.2 mL TubesEpiCypherCatalog #10-0009
10x Magnetic Separator10x GenomicsCatalog #120250/ 230003
Safety warnings
All standard institutional precautions and procedures should be taken when working with and disposing of biological samples and chemical reagents.
Ethics statement
The procedure for use of animals for this experiment must be approved by your institution's Animal Care and Use Committee (ACUC) or equivalent ethics committee(s) PRIOR to conducting any experiments.
Before start
Before starting the protocol, ensure that you have:
New aliquot(s) of frozen spermidine that has never been thawed.
- Spermidine that has been through multiple freeze-thaw cycles and/or has not been exclusively on ice when thawed may not work, resulting in total sample loss.
- I prepared 1 mL of 1 Molarity (M) spermidine and On ice , aliquoted 10 µL each into PCR tubes which were then immediately frozen. I take out tubes as needed and thaw On ice .
Prepared the XLS-version of buffers for fixed samples. See Materials tab for complete recipes.
EDTA-free protease and/or phosphatase inhibitors.
- The MNase cleavage is calcium-dependent, thus EDTA will inhibit.
- A strong magnetic separator. Magnets used for Dynabeads may be insufficient to effectively pull-down beads. We use the 10X Genomics 10X Magnetic Separator HT (part no: 1000394).
- If you are unsure your magnet will work, test a small amount of Concanavalin A beads diluted in Bead Activation Buffer and try pipetting off the buffer and washing the beads with more Bead Activation buffer (10 µL buffer to 1 µL beads). If you keep pulling up beads upon aspiration or the beads are not being pulled to the magnet within a minute or two, you likely need a stronger magnet.
High-quality sample. This is critical, because if the cells are not well-stimulated and not prepared quickly and correctly, the nuclei will disintegrate when you mix with the ConA beads.
A brightfield microscope. You will need to visually inspect nuclei using trypan blue and a hemocytometer to check for integrity, so an automated cell counter may not be sufficient.
Filter pipet tips. Since we want to minimize any cross contamination across samples, the use of filter tips is highly recommended for all steps involving transfer of sample or reagents (i.e., primers, buffer for resuspending beads, etc.) Filter tips are not necessary for ConA bead washing steps during CUT&RUN, but change tips after each wash/disposal of buffer. Similarly, filter tips are not necessary for washes of SPRI beads as the washes are in ethanol.
B cell Isolation and Stimulation: B cell Isolation
B cell Isolation and Stimulation: B cell Isolation
40m
40m
Sacrifice mouse and remove spleen in tissue culture hood.
Note
All following steps in this section must be under sterile conditions.
Crush spleen through a 70-micron filter with MACS media to make a single cell suspension.
Centrifuge cells at 600 x g, Room temperature, 00:05:00 and remove supernatant.
5m
Resuspend cell pellet in 1 mL MACS media.
Add 5 mL ACK lysis buffer to remove red blood cells.
Centrifuge cells at 600 x g, Room temperature, 00:05:00 and remove supernatant.
5m
Resuspend cell pellet in 1 mL MACS media.
Count cells.
Add αCD23 magnetic beads according to number of total cells (100 µL per 100 million cells).
Chill sample at4 °C for 00:15:00 -00:20:00 .
20m
About 00:05:00 before end of incubation in go to step #10 , set up the LS column in the magnetic holder and wash with 7 mL MACS media. Discard flow-through.
5m
Pipet cells+beads directly onto the top of the column.
Once all sample is in the column, add 7 mL MACS media to wash. Discard flow-through.
Remove column from magnet and place into a 15-mL Falcon tube for collection.
Add 7 mL MACS media. Let half of the volume flow through by gravity, and then insert the plunger and firmly but gently, push the remaining sample through the column.
Expected result
This flow through is your purified follicular B cells.
Centrifuge cells at 600 x g, Room temperature, 00:05:00 and remove supernatant.
5m
Resuspend cells in 1 mL of MACS media or B cell media (see next section) and count cells.
B cell Isolation and Stimulation: B cell stimulation
B cell Isolation and Stimulation: B cell stimulation
2d
2d
Note
- A starting recommendation is to prepare more cells than you think you need, as it is better to have more sample to work with in case of technical issues.
- Plan for 2-3x expansion of cells with stimulation.
- You will lose cells if you freeze/thaw cells (about 40-80% yield) and again in the nuclei isolation step (about 60-90% yield).
Note
All following steps in this section must be under sterile conditions.
Prepare the B cell media:
Using pre-warmed RPMI 1640 as the starting buffer, supplement it with:
- 10% FBS
- 1% Glutamax
- 1% Pen/Strep
- 53 micromolar (µM) beta-mercaptoethanol
Sterile filter.
Note
Media must be prepared same day as use for best results.
Add 250,000 cells per mL of media for stimulation.
Add 2 μg/ml αCD40 antibody and 10 ng/ml IL-4.
Aliquot cells into culture plate(s) and/or flask(s).
Aliquot 1 mL -2 mL of culture into a separate plate for day 3 analysis of CSR to make sure your stimulation was successful. Instead of removing plate at 48 hours, remove at 72 hours and then stain for viability, IgG1, and IgM and perform flow cytometry analysis.
Incubate cultures at 5% CO2 and 37 °C for 48:00:00 .
2d
B cell Isolation and Stimulation: Sample collection (directly from culture)
B cell Isolation and Stimulation: Sample collection (directly from culture)
10m
10m
Remove culture from incubator and transfer into a Falcon tube.
Centrifuge at 600 x g, Room temperature, 00:05:00 and discard supernatant.
5m
Resuspend cell pellet in 1 mL of Room temperature DPBS and then dilute up to 10 mL .
Count cells.
Centrifuge cells once more at 600 x g, Room temperature, 00:05:00 . Discard supernatant.
5m
If using fresh cells, proceed directly to Nuclei preparation.
If freezing cells, resuspend pellet with 100% FBS and add DMSO to a final concentration of 10% v/v and aliquot into pre-labeled cryo-safe vials.
- Immediately place in Mr. Frosty slow freezer with isopropanol and place at -80 °C . Once frozen, you can remove tubes from Mr. Frosty and place in a standard freezer box.
Note
We recommend 5-10 million cells per mL, 1 mL per vial.
B cell Isolation and Stimulation: Thawing frozen samples
B cell Isolation and Stimulation: Thawing frozen samples
10m
10m
Remove cryo-safe vial(s) from -80 °C and immediately place in 37 °C water bath/incubator/heat block to quick thaw.
Once thawed, centrifuge at 600 x g, Room temperature, 00:05:00 and carefully pipet off supernatant, being careful to not disturb the pellet.
Note
If your centrifuge does not fit cryovials, try placing the whole cryovial into a 14 mL Falcon tube(or equivalent) and then centrifuging. Use forceps to gently pull out the tube and aspirate off supernatant.
5m
Resuspend pellet with 1 mL DPBS and transfer to 15 mL Falcon tube.
Add DPBS up to 10 mL .
Centrifuge at 600 x g, Room temperature, 00:05:00 and discard supernatant. (1/3)
5m
Resuspend pellet with 1 mL DPBS and take a small aliquot to count cells using Trypan Blue.
Add DPBS up to 10 mL .
Centrifuge at 600 x g, Room temperature, 00:05:00 and discard supernatant. (2/3)
5m
Resuspend pellet with 1 mL DPBS. Then add DPBS up to 10 mL .
Centrifuge at 600 x g, Room temperature, 00:05:00 and discard supernatant. (3/3)
5m
Sample Preparation: Nuclei preparation
Sample Preparation: Nuclei preparation
51m
51m
Note
- It is important that sample preparation be timely with minimal time between steps. Make sure all reagents are prepared before proceeding. See supplemental file for buffer recipes.
- Fixing of cells and then nuclei preparation gives higher-quality sample than fixation of nuclei.
- Do not freeze nuclei – recovery and quality was extremely poor across multiple methods we tested.
Resuspend pellet with 100 µL of dead cell removal magnetic beads (or more if you have more than 10 million cells – refer to kit instructions).
Incubate at Room temperature for 00:15:00 .
15m
About 00:05:00 before incubation ends in go to step #40 , set up an LS column in the magnetic holder and wash with 3 mL DPBS. Discard flow-through.
5m
Place a 15 mL Falcon tube under the LS column (while on the magnet).
Note
You are collecting flow-through so this is critical.
Add DPBS to the sample for a final volume of 500 µL , and apply to the column.
Once all sample is in the column, add 6 mL -7 mL DPBS and let it flow through. (1/2)
Add 6 mL -7 mL DPBS and let it flow through. (2/2)
Note
Your total volume should be 12.5 mL -14.5 mL .
Count and calculate total number of live cells.
Calculate how much formaldehyde is needed to add to your sample to get to a final concentration of 0.1% and have 2.5 Molarity (M) glycine at hand for go to step #49 .
Note
For a 37% stock of formaldehyde you will use 2.7 µL/mL of your sample.
Add formaldehyde to 0.1% and immediately start a timer for 00:01:00 . Close the tube and continuously invert to mix (gently, not shaking).
Note
It is recommended to fix one sample at a time due to the very short time frame for fixation. This ensures all samples are fixed for the correct length of time and evenly mixed with care.
1m
After 60 seconds, immediately add glycine to a final concentration of 125 millimolar (mM) (1:20 dilution) and mix well by gentle inversions.
Centrifuge sample at 600 x g, Room temperature, 00:05:00 and discard supernatant in appropriate and labeled waste container for fomaldehyde.
Note
If working with a small number of cells (<1 million), very carefully aspirate off the supernatant instead of pouring off. This ensures minimal sample loss. This should be the case for all steps moving forward.
5m
Resuspend pellet with 1 mL DPBS. (1/3)
Then, add DPBS up to 10 mL . (1/3)
Centrifuge at 600 x g, Room temperature, 00:05:00 and discard supernatant in appropriate and labeled waste container for fomaldehyde. (1/3)
5m
Resuspend pellet with 1 mL DPBS. (2/3)
Then, add DPBS up to 10 mL . (2/3)
Centrifuge at 600 x g, Room temperature, 00:05:00 and discard supernatant in appropriate and labeled waste container for fomaldehyde. (2/3)
5m
Resuspend pellet with 1 mL DPBS. (3/3)
Then, add DPBS up to 10 mL . (3/3)
Centrifuge at 600 x g, Room temperature, 00:05:00 and discard supernatant in appropriate and labeled waste container for fomaldehyde. (3/3)
5m
Add cold Nuclear Isolation Buffer at 1 mL per 5 million cells, minimum 1 mL.
Note
Do not pipet to mix – instead, gently vortex to reduce sample loss.
Incubate On ice for 00:10:00 .
10m
Centrifuge at 600 x g, 4°C, 00:05:00 and discard supernatant by careful aspiration. (1/2)
5m
Resuspend in 1 mL cold Wash-XLS buffer and then add up to 5 mL . Take a small aliquot, stain with trypan blue, and count nuclei while assessing quality. Sample will likely have scattered cell debris (appearance of dark blue fluff), but that is okay.
Centrifuge at 600 x g, 4°C, 00:05:00 and discard supernatant by careful aspiration. (2/2)
5m
Resuspend nuclei at 1-5 million nuclei per 1 mL in cold Wash-XLS buffer.
Again, take a small aliquot, stain with trypan blue and count nuclei for verification. There should be minimal to no cell debris left, but if it is still extensive, repeat wash step once more go to step #61 , resuspend nuclei go to step #62 , and recount. It is important that your sample is clear of debris to prevent clumping of samples during CUT&RUN.
Note
Prepared nuclei should always remain On ice or at 4 °C .
Sample Preparation: Test nuclei stability with Concanavalin A beads (optional)
Sample Preparation: Test nuclei stability with Concanavalin A beads (optional)
30m
30m
Note
- The purpose of this section is to ensure sample will be able to withstand the CUT&RUN protocol, and it is important that this is done before beginning the experiment. By first preparing a small amount of beads and nuclei, there is no waste of ConA beads in case of sample failure.
- This section can be skipped when you determine your handling of samples to consistently yield stable nuclei.
Vortex Concanavalin A (ConA) beads to resuspend.
Take 3 µL and add to a tube (use 8-strip tubes from Epicypher supplied in kit).
Add 30 µL of Bead Activation buffer, mix, and place on magnetic separator to clear.
Remove supernatant and add 30 µL of Bead Activation Buffer.
Remove tube from magnet to mix by gentle pipetting and replace on magnetic separator to clear.
Remove supernatant and add 5 µL of Bead Activation Buffer.
Remove tube from magnet to mix by gentle pipetting.
Add 10 µL of prepared nuclei to beads, mix by gentle pipetting, and incubate On ice for 00:15:00 -00:30:00 .
30m
Take an aliquot of nuclei+beads and stain with trypan blue. Observe sample under microscope. Nuclei should still be small, round, bright blue, and intact.
Note
- If you see an excessive amount of blue fluffy debris and abnormally-shaped nuclei, check that your buffers were made using the correct concentrations of reagents.
- You may attempt steps 64-70 again, but without knowing the cause of failure, it is unlikely the sample will be stable on the second attempt and will not withstand the CUT&RUN.
- There are many possible reasons this can happen – the most common tend to be poor stimulation of B cells, spermidine that is not fresh and/or not been kept On ice , taking too long to prepare samples/letting them sit around too long, and inadequate fixation, to name a few.
- Stop now and prepare new samples from the beginning.
CUT&RUN: Binding nuclei to ConA beads
CUT&RUN: Binding nuclei to ConA beads
34m
34m
Vortex beads to resuspend.
Take 11 µL of beads per CUT&RUN sample and transfer to a 1.5 mL microcentrifuge tube.
Note
This volume is the same regardless of how many nuclei you use. This ensures the magnetic pellet size is sufficient for pull down.
Add 100 µL of Bead Activation Buffer per sample and mix. (1/2)
Place tube on a magnetic separator and let clear (about 00:02:00 ). (1/2)
2m
Remove buffer and take tube off magnet. (1/2)
Add 100 µL of Bead Activation Buffer per sample and mix. (2/2)
Place tube on a magnetic separator and let clear (about 00:02:00 ). (2/2)
2m
Remove buffer and take tube off magnet. (2/2)
Resuspend beads in 11 µL Bead Activation Buffer per sample.
Dilute nuclei to desired concentration using Wash-XLS Buffer if desired. You will use 100 µL of nuclei each sample, so if you want to use 500,000 nuclei per sample, do not dilute. If you want to use 100,000 nuclei per sample, dilute nuclei 1 in 5, and so forth.
Note
If not using the whole sample, aliquot what will be used into a separate tube.
Add prepared ConA beads to the nuclei. Mix by gentle pipetting and incubate On ice for 00:30:00 .
30m
Take a small aliquot and stain using trypan blue. Check that nuclei are bright, round, and blue before proceeding.
Aliquot 110 µL of nuclei + beads each into 8-strip tubes.
Note
- Once the beads and nuclei have been bound and aliquoted into sample tubes, for all subsequent steps that involve mixing, we recommend tilting strip tubes horizontally and gently shaking back and forth or gentle vortexing and then quick spin.
- We do not recommend mixing by pipetting since the ConA beads will stick to most pipet tips, including low-retention tips, resulting in sample loss.
Place tubes with nuclei and ConA beads on magnetic separator and let clear (about 00:02:00 ).
2m
Remove supernatant and add 50 µL of Antibody Binding Buffer.
Note
- Do not mix by pipette: mix by tilting strip horizontally, gently shaking the strip back and forth, and then quick spin.
- A multi-channel pipet is highly recommended/helpful.
CUT&RUN: Antibody Binding
CUT&RUN: Antibody Binding
8h
8h
Add 1 µg of antibody to each sample for target of interest, including controls H3k4me3 and IgG.
Note
Some manufactures have recommended concentrations for their antibody clones for CUT&RUN – use those amounts if available instead of 1 µg.
Gently mix samples by shaking horizontally or gentle vortexing. Quick spin to collect samples to bottom of the tube.
Place samples on shaker or thermomixer (set to 800 rpm ) at a 30-45 degree angle from vertical kept at 4 °C Overnight .
- It is important for the tubes to be kept upright at an angle and with motion to ensure beads stay suspended. Do not rotate tubes – the reaction volume is small, and beads that get stuck on the cap will dry out, which will destroy your sample.
- By the morning, most of the beads will have settled and that is normal – tilt the tubes on their side and gently mix by shaking and quick spin. Take note if beads are clumpy – this may be a sign that your nuclei have fallen apart.
- If this happens, take 2 µL -3 µL from one of your samples (take note of which) and add to 8 µL -9 µL trypan blue and check under a microscope. Are the nuclei intact? Is there a lot of blue, fluffy debris? If there are no intact nuclei, the experiment has failed and you should start over.
8h
CUT&RUN: Binding and activation of pAG-MNase
CUT&RUN: Binding and activation of pAG-MNase
2h 40m
2h 40m
Remove tubes from thermomixer. Gently agitate the tubes to resuspend beads and give a quick spin to collect all sample to the bottom of tube.
Note
Use of a metal tube block On ice is very helpful for keeping tubes stable and cold.
Place tubes on magnetic separator and let clear.
Remove supernatant and add 200 µL Wash-XLS Buffer while tubes are still on magnet. You do not need to resuspend the beads, just let the buffer gently wash over the beads. (1/2)
Remove supernatant and add 200 µL Wash-XLS Buffer while tubes are still on magnet. You do not need to resuspend the beads, just let the buffer gently wash over the beads. (2/2)
Remove supernatant and add 50 µL Wash-XLS Buffer. Gently shake horizontally or vortex to mix and quick spin to collect sample to the bottom of tube.
Add 2.5 µL pAG-MNase enzyme to each sample and gently shake horizontally or vortex to mix, and quick spin to collect sample to the bottom of tube.
Incubate tubes On ice for 00:30:00 .
30m
Place tubes on magnetic separator and let clear.
Remove supernatant and add 200 µL Wash-XLS Buffer while tubes are still on magnet. (1/2)
Remove supernatant and add 200 µL Wash-XLS Buffer while tubes are still on magnet. (2/2)
Remove supernatant and add 50 µL Wash-XLS Buffer. Gently shake or vortex to mix and quick spin to collect sample to the bottom of tube.
Incubate tubes on ice for 00:05:00 to ensure samples are cold.
5m
Add 1 µL 100 millimolar (mM) Calcium Chloride to each sample. Mix and quickly centrifuge. Wrap samples in plastic wrap or place in a sealed bag, and incubate buried in wet ice for 02:00:00 .
Note
Most complete digestion of fixed samples was found with a 2 hour digestion. Digestion at 30 minutes results in a larger proportion of multinucleosome fragments upon library preparation.
2h
Prior to the end of the incubation, prepare the Stop Master Mix by adding 33 µL of Stop Buffer + 1 µL of E. coli Spike-in DNA per 500,000 nuclei for each sample and mix well.
Note
Spike-in should be approximately 1% of total reads. Scale amount of spike-in as needed for total amount of nuclei.
After 2 hours, add 33 µL of Stop Master Mix+spike-in to each sample. Very gently and briefly shake horizontally once to mix and quick spin to collect sample to the bottom of the tube.
Place samples in a thermocycler set to 37 °C for 00:10:00 .
10m
Quick spin samples and then place on magnetic separator to clear.
Collect supernatant and transfer to fresh 8-strip tubes.
CUT&RUN: Post-CUT&RUN DNA purification
CUT&RUN: Post-CUT&RUN DNA purification
8h 23m
8h 23m
Add 1 µL Proteinase K and 1.6 µL of 5% SDS to each sample. Mix and quick spin.
Place samples in a thermocycler set to 55 °C and incubate Overnight to reverse-crosslink and digest protein.
8h
For CUTANA Version 3 kit and older:89 steps
Next day, transfer samples to 1.5 mL microcentrifuge tubes. Add 420 µL DNA Binding Buffer. Mix well and quick spin to collect sample.
Place DNA purification column in a 2 mL collection tube, and transfer each sample to a column.
Centrifuge at max speed (13000 x g -16000 x g ) for 00:01:00 . Discard flow through.
1m
Add 200 µL DNA Wash Buffer to columns. (1/2)
Centrifuge at max speed (13000 x g -16000 x g ) for 00:01:00 . Discard flow through. (1/2)
1m
Add 200 µL DNA Wash Buffer to columns. (2/2)
Centrifuge at max speed (13000 x g -16000 x g ) for 00:01:00 . Discard flow through. (2/2)
1m
Centrifuge once more at max speed (13000 x g -16000 x g ) for 00:01:00 to remove residual ethanol. Discard flow through.
1m
Transfer columns to clean 1.5 mL microcentrifuge tubes.
Add 12 µL DNA Binding Buffer to the center of each column without scratching the resin. Incubate at Room temperature for 00:05:00 .
5m
Centrifuge at max speed (13000 x g -16000 x g ) for 00:01:00 . Discard columns.
1m
Before proceeding to library preparation, quantify yield of DNA from CUT&RUN experiments using a Qubit fluorometer with the 1x dsDNA High Sensitivity Assay Kit. Use 2 µL of each sample and 198 µL of 1x to analyze.
If possible, use this concentration to calculate the total yield of DNA (for the remaining 23 µL -24 µL ).
Note
- Yields will likely be low and you may not get a concentration reading. Ideally, the positive H3K4me3 sample should have more DNA than the negative IgG control, but when using low numbers of nuclei, you may not get a concentration for most or any of the samples when using inputs lower than 250k. This is okay – quality libraries can still be prepared, and adjustments to the library preparation protocol have been added to accommodate this. If you consistently get no reading after many experiments with the same amount of starting input, it is fine to skip this step and proceed directly to library prep so as to not waste any sample.
Library Preparation: End repair, adapter ligation, and U-excision
Library Preparation: End repair, adapter ligation, and U-excision
8h 23m
8h 23m
Thaw samples On ice , if frozen.
Note
- The library preparation should be performed in a PCR hood to minimize contamination.
- Keep all reagents and mixes on ice unless thawing buffers (particularly End Repair buffer).
- Use of a metal tube block On ice is very helpful for keeping tubes stable so they stay cold.
Thaw End Prep Buffer at Room temperature . Mix well and make sure all solids are dissolved, then place On ice . Thaw Adapter for Illumina On ice .
Note
Do not keep the adapter at Room temperature at any time. This will increase the amount of adapter dimer in the sample.
Transfer 5 ng of CUT&RUN fragments to new strip tubes. If you have less than 5 ng of total DNA (by Qubit), then use the entire 25 µL of sample.
Add 0.1x TE buffer to adjust sample volumes to 25 µL (if not already).
Prepare the End Repair Master mix as follows: Combine 4.2 µL End Prep Buffer and 1.8 µL of End Prep Enzyme per reaction. Mix by gentle vortexing and quick spin.
Add 5 µL of End Repair Master Mix to each sample. Mix by gentle vortexing and quick spin.
Place samples in a thermocycler and perform the following (with heated lid >75 °C ):
- Step 1: 20 °C for 00:20:00
- Step 2: 65 °C for 00:30:00
- Hold at 4 °C
Remove tubes and quick spin to collect sample. Place tubes On ice .
Note
The following step using the thermocycler (go to step #138 ) requires the lid to not be heated.
Prepare the Adapter Ligation Master Mix as follows: in a fresh tube, combine 16.5 µL of Ligation Mix and 0.5 µL Ligation enhancer each sample. Mix by gentle vortexing and quick spin, then place On ice .
- Do NOT add Adapter for Illumina to this mixture as the adapters will ligate together.
If first time using reagent, transfer Adapter for Illumina into a fresh tube On ice and add an equal volume of cold 0.1x TE buffer to make a 0.5x stock.
Note
Using less adapter reduces adapter dimer formation – especially when DNA yields are low.
Add 1.25 µL of diluted Adapter for Illumina to each sample.
Then, add 15.5 µL Ligation Master Mix to each sample.
Mix by gentle vortexing and quick spin.
Place samples in a thermocycler and incubate at 20 °C for 00:15:00 without heated lid.
Remove tubes from thermocycler, quick spin samples, and place in Room temperature rack.
Add 1 µL U-Excision Enzyme to each sample.
Mix by gentle vortexing and quick spin.
Place samples in a thermocycler and incubate at 37 °C for 00:15:00 with lid heated >47 °C .
Remove tubes from thermocycler, quick spin samples.
Note
Samples can be stored at -20 °C at this point.
Library Preparation: First DNA cleanup
Library Preparation: First DNA cleanup
8h 23m
8h 23m
Note
- A multichannel pipet and basins are very helpful here.
- If you have more than one 8-strip tubes and your magnetic separator holds only one strip, perform the cleanup one strip at a time to ensure protocol is accurately performed.
Before cleanup, prepare a fresh 85% mixture of ethanol in molecular biology-grade water. You will need ~500 µL per reaction for this first DNA cleanup.
Vortex SPRIselect beads ~00:00:30 to fully resuspend beads.
Add 47.75 µL SPRIselect beads to each sample. Mix by gentle vortexing and quick spin
Incubate samples at Room temperature for 00:05:00 .
Place tubes on magnetic separator and let clear. Remove supernatant.
Add 180 µL of 85% ethanol to samples (still on magnet), remove supernatant, and discard. (1/2)
Add 180 µL of 85% ethanol to samples (still on magnet), remove supernatant, and discard. (2/2)
Quick spin the tube strip and place back on magnetic separator. Remove any residual liquid.
Place the tubes on a rack at Room temperature and leaving lids open, let air dry for about 00:02:00 .
Note
Keep an eye on them – they should have little to no residual ethanol left and the beads will look slightly glossy, but do not let them completely dry (they will lighten in color and appear crumbly/cracked).
Add 12 µL of 0.1x TE buffer to each tube. Vortex to resuspend beads, quick spin, and incubate at Room temperature for 00:02:00 to release DNA fragments.
Place tubes on magnetic separator and let clear. Collect ~10.5 µL of supernatant and transfer to fresh 8-strip tubes, being careful to not pull up any beads even if there is a small volume of buffer left.
Note
Samples can be stored at -20 °C at this point.
Library Preparation: Indexing PCR
Library Preparation: Indexing PCR
8h 23m
8h 23m
Note
- Use of a metal tube block on ice is very helpful for keeping tubes stable and cold.
- Prior to first use, transfer 10 µL of each indexing primer to 8-strip tubes (in order for easy pipetting with multichannel). Add 40 µL of 0.1x TE buffer to each primer to make a 1 in 5 dilution. Be careful to ensure that there is no cross contamination between primers.
Identify what unique combination of indexing primers (i7 and i5) will be used for each sample and take note. The CUT&RUN Library Prep kit comes with a very helpful table on the Quick-Start card for recording which combinations have already been used.
Thaw indexing primers On ice . Quick spin if needed.
Add 1 µL of assigned and diluted i7 primer to each sample.
Add 1 µL of assigned and diluted i5 primer to each sample.
Add 12.5 µL of Hot Start 2x PCR Master Mix to each sample. Total volume should be 25 µL .
Mix by gentle vortexing and quick spin.
Place tubes in a thermocycler with the following PCR steps (with heated lid 105 °C ):
| A | B | C | D | |
| Step 1 | 98°C | 45 seconds | ||
| Step 2 | 98°C | 15 seconds | 16 cycles | |
| 60°C | 10 seconds | |||
| Step 3 | 72°C | 1 minute | ||
| Hold | 4°C |
PCR cycling conditions
Remove tubes from thermocycler and quick spin.
Note
Samples can be stored at -20 °C at this point.
Library Preparation: Second DNA cleanup
Library Preparation: Second DNA cleanup
8h 23m
8h 23m
Note
- This is a multi-step cleanup. You will perform a double-sided SPRIselect beads selection at 0.6-0.95x ratios. This helps remove any excessive adapter dimers and most multinucleosome fragments.
- If you have more than one set of 8-strip tubes and your magnetic separator holds only one strip, I recommend performing the cleanup one strip at a time to ensure protocol is accurately performed.
Prepare fresh 85% ethanol in MB-grade water if needed. You will need about 900 µL -1000 µL for each sample.
Add 25 µL of 0.1x TE to each tube. Total volume should now be 50 µL .
Vortex SPRIselect beads ~00:00:30 to fully resuspend beads.
Add 32.5 µL SPRIselect beads to each sample (equal to 0.6x ratio of beads to sample). Mix by gentle vortexing and quick spin.
Incubate samples at Room temperature for 00:03:00 .
Place tubes on magnetic separator and let clear. Collect supernatant and transfer it to fresh 8-strip tubes.
Note
Library is in the supernatant as it is excluded from the beads due to the low ratio. Do not throw the supernatant away!
Add 15 µL SPRIselect beads to each sample (equal to 0.35x, bringing total added to 0.95x). Mix by gentle vortexing and quick spin.
Incubate samples at Room temperature for 00:05:00 .
Place tubes on magnetic separator and let clear. Remove supernatant.
Add 180 µL of 85% ethanol to samples (still on magnet), remove supernatant, and discard. (1/2)
Add 180 µL of 85% ethanol to samples (still on magnet), remove supernatant, and discard. (2/2)
Quick spin the tube strip and place back on magnetic separator. Remove any residual liquid.
Place the tubes on a rack at Room temperature and leaving lids open, let air dry for about 00:00:30 -00:01:00 .
Note
Keep an eye on them – they should have little to no residual ethanol left and the beads will look glossy, but do not let them completely dry (they will lighten in color and appear crumbly/cracked). They will dry very quickly due to the low volume of beads.
Add 31.5 µL of 0.1x TE buffer to each tube. Vortex to resuspend beads, quick spin, and incubate at Room temperature for 00:05:00 to release DNA fragments.
Place tubes on magnetic separator and let clear.
Collect 30 µL supernatant and transfer to fresh 8-strip tubes, being careful to not pull up any beads even if there is a small volume of buffer left.
Note
DNA can be stored at -20 °C until ready to perform sequencing.
Sequencing: Library QC
Sequencing: Library QC
8h 23m
8h 23m
Note
Before proceeding to sequencing, check libraries by both Qubit (for concentration) and Bioanalyzer (for fragment size distribution):
For Qubit, use 1 µL -2 µL of library with 1x dsDNA high sensitivity reagent up to 200 µL .
For Bioanalyzer, use Agilent High Sensitivity DNA kit. Follow manufacturers protocol.
Using these results, calculate the molarity and/or moles of each library.
Sequencing
Sequencing
8h 23m
8h 23m
Determine which sequencing kit you will use for your samples.
Note
For sequencing, paired end and minimum 50 bps is needed to map data. Depending on the target, 3-5 million reads may be enough, but if this is your first time running a target (especially non-histone), then plan to sequence 10-15 million reads each sample.
Pool libraries into equimolar ratios.
- Prepare an equimolar mixture of libraries for sequencing, calculated by sequence-able fragment sizes (~500 bps and under).
- If needed, perform one more SPRIselect bead purification on library pool to remove any remaining adapter dimer and/or multinucleosome fragments.
- Reanalyze using Qubit and Bioanalyzer
Sequence using an appropriate sequencing kit.
Sequencing: Data Analysis
Sequencing: Data Analysis
8h 23m
8h 23m
Copy raw data files from the sequencer to your computer.
Note
It's always a good idea to have backups of the data as well.
Using bcl2convert (Illumina), perform demultiplexing.
Using Trim Galore!, trim adapters off the reads.
- Options --paired --fastqc --fastqc_args "--nogroup"
Using bowtie2, align reads to the appropriate genome (mm39, hg38, etc.).
- Mode -x, -1 and -2 flags for paired sample files.
Using SAMtools, perform the function collate.
Using SAMtools, perform the function fixmate.
- Options -rm
Using SAMtools, perform the function sort.
- Options -@ 8 -T $TMPDIR
Using SAMtools, perform the function markdup.
- Options -r -T $TMPDIR
Using MACS2, perform the function callpeak.
- Options -t for sample, -c for IgG control, -B
Using Integrative Genome Browser, input your sequence (.fa) and annotated gene files (.gff or similar) for genome of choice.
Using Integrative Genome Browser, input -pileup.bdg peak files.
Data can now be visualized by chromosome and gene.
Additional analyses may be performed as appropriate, such as IDR, motif analysis, etc.
Protocol references
Citations:
This protocol has been adapted with Epicypher CUTANA ChIC/CUT&RUN (SKU: 14-1048) and CUTANA Library prep kits (SKU: 14-1001 & SKU: 14-1002) and protocols along the original protocols published by Skene and Henikoff.
1. Skene, P. & Steven Henikoff, S. An efficient targeted nuclease strategy for high-resolution mapping of DNA binding sites eLife 6:e21856 (2017). https://doi.org/10.7554/eLife.21856
2. Skene, P., Henikoff, J. & Henikoff, S. Targeted in situ genome-wide profiling with high efficiency for low cell numbers. Nat Protoc 13, 1006–1019 (2018). https://doi.org/10.1038/nprot.2018.015