TITLE: CUT&amp;RUN abbreviated protocol v2
AUTHORS: CG -

[DESCRIPTION]
An abbreviated version of Henikoff/Janssens Cut&RUN protocol. We use in-house prepared pAG-MNase and standard conditions. If library quality is poor, consider using one of the high-salt or low-salt conditions offered in the original protocol. 

[BEFORE_START]
REAGENT SETUP
 
5% Digitonin To reconstitute enough digitonin for an experiment, weigh out the powder in a 2 mL microcentrifuge tube, boil water in a small beaker in a microwave oven, and pipette in and out to warm the 1000 μL pipette tip. Pipette the hot water into the tube with the digitonin powder to make 5% (w/v), close the cap and quickly vortex on full until the digitonin is completely dissolved. If refrigerated, this stock can be used within a week, but will need reheating as the digitonin slowly precipitates. The effectiveness of digitonin varies between batches, so testing permeability of Trypan blue is recommended to determine the concentration to use for a cell type. We have obtained excellent results for K562 cells with 0.02-0.1% digitonin.
NOTE: The 5% digitonin stock may also be prepared by dissolving in dimethylsulfoxide (DMSO), and can then be stored at -20 °C for up to 6 months. Be aware that DMSO can absorb through the skin.
CAUTION: Digitonin is toxic and care should be taken especially when weighing out the powder. 
 
Binding buffer: Mix 20 mL of Binding Buffer in a 50 mL conical tube. Store the buffer at 4 °C for up to 6 months.  
 
 
Activate Concanavalin A-coated beads in Binding Buffer: Gently resuspend and withdraw enough of the slurry such that there will be 10 μL for each final sample and/or digestion time point. Transfer into 1.5 mL Binding buffer in a 2 ml tube. Place the tube on a magnet stand to clear (30 s to 2 min). Withdraw the liquid, and remove from the magnet stand. Add 1.5 mL Binding buffer, mix by inversion or gentle pipetting, remove liquid from the cap and side with a quick pulse on a microcentrifuge. Resuspend in a volume of Binding buffer equal to the volume of bead slurry (10 μL per final sample).
Wash buffer: Mix 50 mL of Wash Buffer. This buffer can be stored at 4 °C for up to 1 week, however, Roche Complete Protease Inhibitor tablet should be added fresh on the day of use.
NOTE: A concentration of salt that is in the physiological range avoids stress when washing the cells and mixing with beads. Spermidine in the wash buffer is intended to compensate for removal of Mg2+ during incubation in the Antibody Buffer, which might otherwise affect chromatin properties.

  
 
 Dig-wash buffer: Mix 150-600 µL 5% (wt/vol) digitonin with 30 mL Wash Buffer for a final concentration of digitonin between 0.025% and 0.1% (wt/vol). Store this buffer on ice or at 4 °C for up to 1 day, and vortex before use.
NOTE: The effectiveness of digitonin varies between batches, so testing for full permeability of Trypan blue is recommended to determine the concentration to use for a cell type. We have obtained excellent results for H1 and K562 cells with 0.05% digitonin (300 µL 5% (wt/vol) digitonin in 30 mL Wash Buffer). For simplicity, we use this same buffer for all steps starting from the incubation in primary antibody until the chromatin digestion.

Antibody buffer: Mix 8 μL 0.5 M EDTA with 2 mL Dig-wash buffer and place on ice.
NOTE: The presence of EDTA during antibody treatment removes excess divalent cations used to activate the Concanavalin A-coated beads, as well as endogenous cations from the cells of interest. This serves to halt metabolic processes, stop endogenous DNAse activity, and prevent carry-over of Ca2+ from the Binding Buffer that might prematurely initiate strand cleavage after addition of pA-MNase. Washing out the EDTA before pA-MNase addition avoids inactivating the enzyme.


2X STOP Buffer: Mix 5 mL of 2X STOP Buffer. Store the buffer at 4 °C for up to 1 week.
 
 




 




[GUIDELINES]
The protocol workflow is as follows:
   
Day 1, Cells to DNA
Binding cells to beads (Steps 1-8, 30 min)  
Permeabilize cells and bind primary antibodies (Steps 9-13, 2.5 hr–overnight, longer incubations provide higher yields) 
Bind secondary antibody as required (Steps 14-20, 15 min-1.5 hr) 
Bind Protein A-MNase or Protein A/G-MNase fusion protein (Steps 21-26, 1.5 hr) 
Chromatin Digestion and Release: Standard CUT&RUN (Steps 27-37, 1.5 hr)
Phenol Chloroform Extraction (Steps 57-67, 1.5 hr)
 
Days 2-4, Library preparation and sequencing
Sample Analysis Pre-Library Prep (optional) (Steps 68-69, 1 hr)  
End Repair and Adapter Ligation (Steps 70-78, 3 hr–overnight)
PCR Enrichment of CUT&RUN Libraries (Steps 79-109, 2-3 hr)
CUT&RUN Library Analysis and Sequencing (Steps 110-112, variable timing)

Day 5
Data processing and analysis (Steps 113-114, variable timing) 
 
 

 
EQUIPMENT
 
Centrifuge Eppendorf 5810, swinging bucket
Centrifuge Eppendorf 5424, fixed angle rotor
Centrifuge Eppendorf 5415R, refrigerated fixed angle rotor
Macsimag magnetic separator (Miltenyi, cat. no. 130-092-168), which allows clean withdrawal of the liquid from the bottom of 1.7 and 2 ml microfuge tubes.
Vortex mixer (e.g., VWR Vortex Genie)
Micro-centrifuge (e.g., VWR Model V)
1.5-ml microcentrifuge tubes (Genesee, cat. no. 22-282)
2-ml microcentrifuge tubes (Axygen, cat. no. MCT-200-C)
Tube rotator (Labquake, Thermo Fisher)
Heater block with wells for 1.5-ml microcentrifuge tubes
Water baths (set to 37°C and 70 °C)
MaXtract phase-lock microcentrifuge tubes (Qiagen, cat. no. 139046)
Capillary electrophoresis instrument (e.g. Agilent Tapestation 4200)
Qubit Fluorometer (Life Technologies, cat. no. Q33216)
 
INTRODUCTION
 
Experimental Design
 
The CUT&RUN method for the in situ targeted cleavage and release of chromatin complexes is straightforward and can be completed in under a day using standard lab equipment. Here we provide a detailed CUT&RUN protocol that now includes various optional modifications to the MNase digestion reaction that can be used to improve data quality or increase throughput in specific situations. One of the strengths of CUT&RUN is that the entire reaction is performed in situ, whereby the antibody and pA- or pA/G-MNase are free to diffuse into the nucleus. The original protocol used nuclei prepared by a combination of hypotonic lysis and treatment of cells with Triton X-100. This has been successful with a number of cell lines, but we have recently adapted the protocol to use cells permeabilized by the non-ionic detergent digitonin, which has been successfully used in other in situ methods, including ChEC-seq and ATAC-seq. Digitonin partitions into membranes and extracts cholesterol. Membranes that lack cholesterol are minimally impacted by digitonin. Nuclear envelopes are relatively devoid of cholesterol compared to plasma membranes. As such, treatment of cells with digitonin represents a robust method for permeabilizing cells without compromising nuclear integrity. The protocol described here uses digitonin, but it is possible that individual experimental situations call for generating intact nuclei by other means, and such nuclei can be prepared by a suitable method, bound to concanavalin A-coated beads as per the protocol provided in Skene and Henikoff (eLife, 2017), and then enter the protocol below at step 9.
One of the limitations of a protocol that has inherently low background and is amenable to low cell numbers is that the amount of DNA recovered can be very low, such that analysis even by sensitive capillary electrophoresis or picogreen assays (e.g. Agilent Tapestation and Qubit) are problematic. In addition, high resolution mapping techniques that cleave a minimal footprint are not suitable to PCR-based analysis of known binding loci, as it is not commonly possible to design ~50 bp PCR amplicons. As such, we recommend using a positive control antibody that targets an abundant epitope and therefore the DNA can be readily detected. We have successfully used a rabbit monoclonal antibody raised against H3K27me3, with capillary electrophoresis showing with the amount of cleaved fragments being proportional to the number of starting cells. A nucleosomal ladder is expected by Tapestation or other sensitive electrophoretic analysis method (Fig. 2), and the use of a monoclonal antibody avoids potential lot-to-lot variation that can complicate troubleshooting. For less abundant epitopes, including many transcription factors, it is harder to detect the cleaved fragments by even sensitive electrophoretic analysis (Supplementary Figure 1). Once the expected digested DNA pattern is observed for the positive control by capillary electrophoresis such as H3K27me3, it is not necessary to sequence this sample. As a negative control, we recommend the use of a non-specific rabbit IgG antibody that will randomly coat the chromatin at low efficiency without sequence bias. We do not recommend a no-antibody control, as the lack of tethering increases the possibility that slight carry-over of pA-MNase will result in preferential fragmentation of hyper-accessible DNA.
 

 


 

 
In our previously published study, we showed that targeted cleavage occurred within seconds of adding Ca2+ ions, and by virtue of being a sterically regulated tethered reaction, the cleavage pattern was constant over time. However, longer digestion times release more material with no apparent change in the signal-to-noise ratio (Supplementary Figure 2). We therefore recommend digesting for 30 minutes as a starting point that can be tailored based upon epitope abundance and antibody concentration.
 
 
Limitations
 
As is the case with ChIP, the success of CUT&RUN depends in large part on the affinity of the antibody for its target and its specificity under the conditions used for binding. Because antibodies bind to their epitopes in the solid state using CUT&RUN, we would expect that antibodies successfully tested for specificity by immunofluorescence (IF) would be likely to work with CUT&RUN, with the caveat that IF generally involves fixation, whereas formaldehyde fixation decreases the efficiency of CUT&RUN.
In the standard CUT&RUN protocol we recommend allowing the cleaved chromatin complexes to diffuse out of the nuclei, thereby permitting simple isolation of the cut DNA from the supernatant fraction with the undigested genome retained in the intact nuclei. However, it is possible that a chromatin complex is too large to diffuse out or that protein-protein interactions retain the cleaved complex. In such cases, total DNA may be extracted after the digestion. By doing a very simple negative size selection using paramagnetic carboxylated beads (e.g. Agencourt AMPure XP beads) large genomic DNA can be removed prior to preparing CUT&RUN sequencing libraries. In Skene and Henikoff (eLife, 2017) we showed that this strategy was successful for the ~1 MDa yeast RSC complex.
TROUBLESHOOTING
 


[STEPS]
SECTION: Binding cells to beads (~30 min)
1. 
Harvest fresh culture(s) at room temperature and count cells. The same protocol can be used for up to 500,000 mammalian cells per sample and/or digestion time point.
SECTION: Binding cells to beads (~30 min)
2. Centrifuge 3 min 600 x g at room temperature and withdraw liquid.
3 min
SECTION: Binding cells to beads (~30 min)
3. Resuspend in 1.5 mL room temperature Wash buffer by gently pipetting and transfer if necessary to a 2 mL tube.
 
1.5 2
SECTION: Permeabilize cells and bind primary antibodies (2.5 hours - overnight)
10. Place on the magnet stand to clear and pull off the liquid.

SECTION: Permeabilize cells and bind primary antibodies (2.5 hours - overnight)
11. Place each tube at a low angle on the vortex mixer set to low (~1100 rpm) and squirt 50-150 μL of the Antibody buffer per sample along the side while gently vortexing to allow the solution to dislodge most or all of the beads. Tap to dislodge the remaining beads150 µL
SECTION: Permeabilize cells and bind primary antibodies (2.5 hours - overnight)
13. Place on a nutator or tube rotator at room temperature for ~2 hr or at 4 °C overnight.
120 min

SECTION: Bind Protein A-MNase or Protein A/G-MNase fusion protein (1.5 hours)
18. Place each tube at a low angle on the vortex mixer set to low (~1100 rpm). Squirt 150 μL of the Protein A-MNase or Protein A/G-MNase fusion protein at 700 ng/mL (e.g., 1:200 of a 140 µg/mL glycerol stock) in Dig-wash buffer (per sample and/or digestion time point) along the side while gently vortexing to allow the solution to dislodge most or all of the beads. Tap to dislodge the remaining beads.
150 1
SECTION: Bind Protein A-MNase or Protein A/G-MNase fusion protein (1.5 hours)
19. Place on the nutator or tube rotator at 4 °C for ~1 hr.
 
60 min
SECTION: Phenol Chloroform Extraction (~1.5 hours)
31. To each sample add 2 μL 10% SDS (to 0.1%), and 2.5 μL Proteinase K (20 mg/ml). Mix by inversion and incubate 1 hr @ 50 °C.
 
2 1
2.5 1
50 10
60 min

SECTION: Phenol Chloroform Extraction (~1.5 hours)
32. Add an equal volume of Phenol Chloroform to the sample (e.g. to 200 µL sample add 200 µL Phenol Chloroform). Mix by full-speed vortexing ~2 s.
 
200 1
2 s
SECTION: Phenol Chloroform Extraction (~1.5 hours)
33. Transfer to a phase-lock tube (e.g., Qiagen MaXtract), and centrifuge 5 min, room temperature @ 16,000 x g.
 
5 min
SECTION: Phenol Chloroform Extraction (~1.5 hours)
34. Add an equivalent volume of chloroform to the initial sample volume (e.g. for a 200 µL starting sample volume add 200 µL chloroform). Invert ~10X to mix and then centrifuge 5 min, room temperature @ 16,000 x g.
 
200 1
5 min
SECTION: Phenol Chloroform Extraction (~1.5 hours)
35. Remove the top liquid phase by pipetting to a fresh tube containing 2 μL 2 mg/mL glycogen (diluted 1:10 from 20 mg/mL glycogen stock).
 
2 1
SECTION: Phenol Chloroform Extraction (~1.5 hours)
36. Add 500 μL 100% ethanol and mix by vortexing or tube inversion.
 
500 1
SECTION: Phenol Chloroform Extraction (~1.5 hours)
37. Chill on ice and centrifuge 10 min, 4 °C @ 16,000 x g.
 
10 min
4 10
SECTION: Phenol Chloroform Extraction (~1.5 hours)
38. Pour off the liquid and drain on a paper towel.
SECTION: Phenol Chloroform Extraction (~1.5 hours)
39. Rinse the pellet in 1 ml 100% ethanol and centrifuge 1 min, 4 °C @ 16,000 x g.
 
1 2
1 min
4 10
SECTION: Phenol Chloroform Extraction (~1.5 hours)
40. Carefully pour off the liquid and drain on a paper towel. Air dry.
 5 min

SECTION: Phenol Chloroform Extraction (~1.5 hours)
41. When the pellet is dry, dissolve in 30-50 μL 1 mM Tris-HCl pH 8 0.1 mM EDTA, then transfer to a new 0.6 mL Lo-Bind microcentrifuge tube.
30 1
SECTION: CUT&amp;RUN Library Analysis and Sequencing
42. Quantify library yield using dsDNA-specific assay, such as Qubit.
SECTION: CUT&amp;RUN Library Analysis and Sequencing
43. Determine the size distribution of libraries by Agilent 4200 TapeStation analysis.
SECTION: CUT&amp;RUN Library Analysis and Sequencing
44. Pool samples at equimolar concentrations and perform paired-end Illumina sequencing on the barcoded libraries following the manufacturer’s instructions.

SECTION: Data Processing and Analysis
45. We align paired-end reads using Bowtie2 version 2.2.5 with options: --local --very-sensitive- local --no-unal --no-mixed --no-discordant --phred33 -I 10 -X 700. For mapping spike-in fragments, we also use the --no-overlap --no-dovetail options to avoid cross-mapping of the experimental genome to that of the spike-in DNA.

 


SECTION: Data Processing and Analysis
46. Scripts are available from https://github.com/Henikoff/Cut-and-Run for spike-in calibration and for peak-calling.

SECTION: Binding cells to beads (~30 min)
4. Centrifuge 3 min 600 x g at room temperature and withdraw liquid.
 
3 min
SECTION: Binding cells to beads (~30 min)
7. While gently vortexing the cells at room temperature, add the ConA-coated magnetic bead slurry.
SECTION: Binding cells to beads (~30 min)
8. Rotate 5-10 min at room temperature.
 
10 min
SECTION: Binding cells to beads (~30 min)
5. Repeat steps 3 and 4 two more times.
   

 

SECTION: Binding cells to beads (~30 min)
6. Resuspend in 1 mL room temperature Wash Buffer by gently pipetting.
SECTION: Permeabilize cells and bind primary antibodies (2.5 hours - overnight)
9. Mix well by vigorous inversion to ensure the bead-bound cells are in a homogenous suspension and divide into aliquots in 1.5-mL tubes, one for each antibody to be used.
 

SECTION: Permeabilize cells and bind primary antibodies (2.5 hours - overnight)
12. Mix in the primary antibody to a final concentration of 1:100 or to the manufacturer’s recommended concentration for immunofluorescence.
SECTION: Bind Protein A-MNase or Protein A/G-MNase fusion protein (1.5 hours)
17. Remove liquid from the cap and side with a quick pulse on a micro-centrifuge. Place on the magnet stand to clear (~30 s) and pull off all of the liquid.
 
30 s
SECTION: Bind Protein A-MNase or Protein A/G-MNase fusion protein (1.5 hours)
14. Remove liquid from the cap and side with a quick pulse on a micro-centrifuge. Place on the magnet stand (~30 sec) to clear and pull off all of the liquid.
 30 s
SECTION: Bind Protein A-MNase or Protein A/G-MNase fusion protein (1.5 hours)
15. Add 1 mL Dig-Wash buffer, mix by inversion, or by gentle pipetting if clumps persist.
1 mL
SECTION: Bind Protein A-MNase or Protein A/G-MNase fusion protein (1.5 hours)
16. Repeat Dig-wash steps 21-22.
   
SECTION: Chromatin Digestion and Release Option 1: Standard CUT&amp;RUN (1.5 hours)
24. Place each tube at a low angle on the vortex mixer set to low (~1100 rpm) and add 100 μL of the Dig-wash buffer (per sample and/or digestion time point) along the side while gently vortexing to allow the solution to dislodge most or all of the beads. Tap to dislodge the remaining beads.
100 µL
SECTION: Chromatin Digestion and Release Option 1: Standard CUT&amp;RUN (1.5 hours)
20. Remove liquid from the cap and side with a quick pulse on a micro-centrifuge. Place on the magnet stand (~30 s) to clear and pull off all of the liquid.
30 s
SECTION: Chromatin Digestion and Release Option 1: Standard CUT&amp;RUN (1.5 hours)
21. Add 1 mL Dig-wash buffer, mix by inversion, or by gentle pipetting if clumps persist.
1 mL
SECTION: Chromatin Digestion and Release Option 1: Standard CUT&amp;RUN (1.5 hours)
22. Repeat Dig-wash steps 27-28.
   
SECTION: Chromatin Digestion and Release Option 1: Standard CUT&amp;RUN (1.5 hours)
25. Insert tubes into the 1.5 mL wells of a heater block sitting in wet ice to chill down to 0 °C.
0 °C
SECTION: Chromatin Digestion and Release Option 1: Standard CUT&amp;RUN (1.5 hours)
26. Remove each tube from the block, mix in 2 μL 100 mM CaCl2 (per sample and/or digestion time point), diluted 1:10 from a 1 M stock, with gentle vortexing and immediately replace the tube in the 0 °C block
2 µL0 °C
SECTION: Chromatin Digestion and Release Option 1: Standard CUT&amp;RUN (1.5 hours)
27. Incubate at 0 °C for the desired digestion time (default is 30 min).
30 min
°C
SECTION: Chromatin Digestion and Release Option 1: Standard CUT&amp;RUN (1.5 hours)
28. Add 100 μL 2X STOP Buffer and mix by gentle vortexing. When there are multiple digestion time points, remove 100 μL and add to a new tube containing 100 μL 2X STOP Buffer and mix by gentle vortexing.
100 µL
SECTION: Chromatin Digestion and Release Option 1: Standard CUT&amp;RUN (1.5 hours)
29. Incubate 30 min @ 37 °C to release CUT&RUN fragments from the insoluble nuclear chromatin.
30 min
37 °C
SECTION: Chromatin Digestion and Release Option 1: Standard CUT&amp;RUN (1.5 hours)
30. Place on the magnet stand to clear. Cleanly transfer the supernatant containing digested chromatin to a fresh 1.5-mL microcentrifuge tube and proceed with Phenol Chloroform Extraction (Step 57).
200 µL

SECTION: Chromatin Digestion and Release Option 1: Standard CUT&amp;RUN (1.5 hours)
23. Remove liquid from the cap and side with a quick pulse on a micro-centrifuge. Place on the magnet stand to clear (~30 s) and pull off all of the liquid.
30 s