Jan 17, 2024

Cleavage Under Targets and Release Using Nuclease (CUT&RUN) V.2

PLOS One
Peer-reviewed method
DOI
https://dx.doi.org/10.17504/protocols.io.x54v9p8ppg3e/v2
  • Geneva Miller1,
  • Lindsey M. Rollosson1,
  • Carrie Saada1,
  • Serenity J. Wade1,
  • Danae Schulz1
  • 1Harvey Mudd College, Claremont, CA
  • PLOS ONE Lab Protocols
  • Spotlight series
Danae Schulz
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DOI: https://dx.doi.org/10.17504/protocols.io.x54v9p8ppg3e/v2
External link: https://doi.org/10.1371/journal.pone.0292784
Protocol Citation: Geneva Miller, Lindsey M. Rollosson, Carrie Saada, Serenity J. Wade, Danae Schulz 2024. Cleavage Under Targets and Release Using Nuclease (CUT&RUN). protocols.io https://dx.doi.org/10.17504/protocols.io.x54v9p8ppg3e/v2Version created by Danae Schulz
Manuscript citation:
Miller G, Rollosson LM, Saada C, Wade SJ, Schulz D (2023) Adaptation of CUT&RUN for use in African trypanosomes. PLOS ONE 18(11): e0292784. https://doi.org/10.1371/journal.pone.0292784
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: January 09, 2024
Last Modified: January 17, 2024
Protocol  Integer ID: 93219
Keywords: Trypanosoma brucei, CUT&RUN, genomic occupancy, mapping genomic protein binding sites, interest in the protozoan parasite trypanosoma brucei, protozoan parasite trypanosoma brucei, protocol for parasite, epitope tag for the protein, parasite, cleavage under target, epitope tag, using nuclease, native protein, protein, nuclease
Abstract
This Cleavage Under Targets and Release Using Nuclease (CUT&RUN) protocol produces genomic occupancy data for a protein of interest in the protozoan parasite Trypanosoma brucei. The data produced is analyzed in a similar way as that produced by ChIP-seq. While we describe the protocol for parasites carrying an epitope tag for the protein of interest, antibodies against the native protein could be used for the same purpose.

The last step contains a supplemental video with extra context and tips, as part of the protocols.io Spotlight series, featuring conversations with protocol authors.
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Guidelines
Guidelines

Pay extra attention to time-sensitive incubation periods, particularly the DNA cleavage step. We find that working through the protocol rather speedily produces the best results, so don’t dawdle.

Additional Notes

We keep our centrifuges at 10 °C to make sure the samples don’t freeze if the temperature drifts slightly. All spin steps of the protocol can be performed anywhere between 4 °C and 10 °C .

While we have not rigorously tested whether increasing the exposure time to saponin influences permeabilization, we recommend that the saponin permeabilization step be completed in smaller batches of samples if large numbers of samples (>10) are being processed simultaneously.

Materials
Materials

  1. Spermidine trihydrochlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #S2501
  2. SaponinMerck Millipore (EMD Millipore)Catalog #558255
  3. Sodium ChlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #S9888
  4. Calcium chlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #C4901
  5. Tris Hydrochloride (Tris-HCl)Merck MilliporeSigma (Sigma-Aldrich)Catalog #RES3098T-B7
  6. cOmplete™ EDTA-free Protease Inhibitor CocktailMerck MilliporeSigma (Sigma-Aldrich)Catalog #11873580001
  7. UltrapPure 0.5M EDTA pH 8.0Invitrogen - Thermo FisherCatalog #15575020
  8. Bioworld EGTA Buffer 0.5M pH 8.0 100mlFisher ScientificCatalog #50-255-957
  9. mouse anti-rabbit IgG-PESanta Cruz BiotechnologyCatalog #sc-3753
  10. Anti-HA antibody produced in rabbitMerck MilliporeSigma (Sigma-Aldrich)Catalog #H6908 , primary antibody will vary depending on your system
  11. Rabbit anti-H3 (gift)
  12. Sodium dodecyl sulfate solutionMerck MilliporeSigma (Sigma-Aldrich)Catalog #428018
  13. Proteinase KThermo Fisher ScientificCatalog #EO0491
  14. Monarch RNase A – 1 ml (2x0.5ml)New England BiolabsCatalog #T3018L
  15. Ampure XP beads Beckman CoulterCatalog #A63881
  16. CUT&RUN pAG-MNase and Spike-In DNACell Signaling TechnologyCatalog #40366

Equipment

  1. Microcentrifuge (Eppendorf 5424R)
  2. Swinging bucket centrifuge (Eppendorf 5920R)
  3. 1.7ml Olympus microtubes (Genesee 22-281)
  4. Olympus 50ml Centrifuge Tubes (Genesee 21-108)
  5. Roto-Mini Plus Variable Speed Rotator with tube holders, 115V (ThermoFisher Scientific, 1159P34)
  6. Novocyte Flow Cytometer 2000
Buffers
NP-S Buffer with 0.1% Saponin
AB
Individual Components
spermidine0.5 mM
Saponin0.1% (vol/vol)
NaCl50 mM
Tris-Cl (pH 7.5)10 mM
Store at 4ºC
Add protease inhibitors just before use.

NP-S Buffer no detergent
AB
Individual Components
spermidine0.5 mM
NaCl50 mM
Tris-Cl (pH 7.5)10 mM
Store at 4ºC
Add protease inhibitors just before use.
Antibody Buffer
NP-S Buffer with 2mM EDTA

2X Stop Buffer
AB
EDTA20mM
EGTA40mM
Add 50 pg of yeast spike-in DNA to each reaction, or 50 pg/100 µl of 2X Stop Buffer.














Before start
Before Start

We use 50-75 million bloodstream parasites per sample. Parasites are cultured in HMI9 media with incubation at 37 °C and 5% CO2. Cultures should be prepared in advance so that sufficient numbers of parasites are available for each sample.

The protocol works best when everything is kept cold prior to cutting with the protein A-MNase fusion protein. We recommend keeping buffers chilled on ice and pre-cooling centrifuges to 4-10 °C . Protease inhibitors should be added to the NP-S buffer just before commencing the experiment. The amount of 2X Stop Buffer required for the experiment should be calculated and yeast spike-in DNA should be added prior to starting (see 2X Stop Buffer recipe below).

Prepare cells
Count parasite cultures with hemocytometer or other preferred counting method.
Spin down cells in centrifuge at 2800 x g, 00:10:00 .
Note
This spin step can be performed at Room temperature or at 10 °C .

10m
Remove supernatant and resuspend in small amount of remaining media (~100 µL ).
If needed, combine samples from multiple Eppendorf tubes so that each final tube has 75 million cells, and spin again at 2800 x g, 10°C, 00:04:00 in microcentrifuge. Remove supernatant.

4m
Permeabilize cells
Wash all samples with 1 mL NP-S Buffer with 0.1% Saponin.

Spin at 4600 x g, 10°C, 00:04:00 . Remove sup.
4m
Primary Antibody Binding
Resuspend each sample in 100 µL NP-S Buffer with 0.1% Saponin.

Add EDTA to 2 millimolar (mM) final (4 µL of 0.05 Mass Percent EDTA/ tube).
Add 5 µg α-HA antibody (or other antibody against protein of interest for experimental sample) OR add 5 µg α-H3 antibody (for control sample, might require titration) OR add 5 µg α-IgG antibody (for control sample).
Incubate for 00:45:00 on tube rotator at Room temperature .

45m
Add 1 mL NP-S Buffer no detergent to each sample.
Spin at 4600 x g, 10°C, 00:04:00 . Remove supernatant.

4m
Add 1 mL NP-S Buffer no detergent to each sample.

Take out aliquot of 1 million cells for flow cytometry analysis (~14 µL if starting with 75 million cells). Set aside On ice .
Spin the remainder of the sample at 4600 x g, 10°C, 00:04:00 . Remove supernatant. (total 2 washes).
4m
pAG-MNase binding
Resuspend each sample in 100 µL of buffer NP-S Buffer no detergent.

Add 1.5 µL of pAG-MNase enzyme to each sample.
Incubate for 00:45:00 at Room temperature on rotator.

45m
FACS Sample preparation
Add 1 mL NP-S Buffer no detergent to 1 million cell aliquot prepared above.
Note
We also routinely use HMI-9 media for the flow cytometry antibody incubation and washes.

Spin at 4600 x g, 00:04:00 .

4m
Resuspend in 100 µL NP-S Buffer no detergent.
Stain with α-rabbit IgG PE at 1:200 for 00:15:00 at Room temperature .
15m
Wash.
Wash in 1 mL NP-S Buffer no detergent (or HMI-9) at 7000 rpm, 00:04:00 . (1/2)

4m
Wash in 1 mL NP-S Buffer no detergent (or HMI-9) at 7000 rpm, 00:04:00 .(2/2)

4m
Resuspend in 300 µL NP-S Buffer no detergent (or HMI-9).
Transfer sample into flow cytometry tube.
Analyze on flow cytometer.
pAG-MNAse wash

Note
continued after 45m incubation above.
Add 1 mL NP-S Buffer no detergent to each sample.
Spin at 4600 x g, 10°C, 00:04:00 . Remove supernatant.

4m
Add 1 mL NP-S Buffer no detergent to each sample.
Spin at 4600 x g, 10°C, 00:04:00 . Remove supernatant.
4m
Targeted Digestion Preparation
Make sure to prepare enough 2X Stopbuffer with spike in control.
Note
This is the section where timing is very important.

Targeted Digestion
Resuspend each sample in 100 µL buffer NP-S Buffer no detergent.

Incubate at Room temperature for 00:05:00 .
5m
Add 2 µL 100 millimolar (mM) CaCl2 to all samples (final concentration = 2mM), mix by flicking.
Incubate all samples at 25 °C for 00:05:00 .
Note
We recommend using a heat block as ambient temperature in the lab can vary.


5m
Add 100 µL 2X Stop buffer to each sample and mix by flicking.
Chromatin Release
Incubate 00:10:00 at 37 °C to release CUT&RUN fragments from the insoluble nuclear chromatin.
10m
Spin at 4600 x g, 10°C, 00:04:00 . Remove supernatant into new tube. SAVE SUPERNATANT.

4m
DNA Extraction
10m
To all samples add 2 µL of 10% SDS (final concentration = 0.1%), 3.3 µL of 10 µL proteinase K (165µg/ml), and 1.33 µL of 1 µL RNAse A (6.5µg/ml).
Mix by gentle flicking and incubate for 00:10:00 at 70 °C .

10m
Purify using Ampure XP beads at 1.8X or phenol chloroform extraction.
Note
Following DNA purification, high-throughput sequencing libraries can be prepared using the preferred method of the research lab.

Spotlight video
Protocol references
Citations

Ashby E, Paddock L, Betts HL, Liao J, Miller G, Porter A, Rollosson LM, Saada C, Tang E, Wade SJ, Hardin J, Schulz D. 2022. Genomic occupancy of the bromodomain protein Bdf3 is dynamic during differentiation of African trypanosomes from bloodstream to procyclic forms. mSphere 7:e00023-22.

Skene PJ, Henikoff S. 2017. An efficient targeted nuclease strategy for high-resolution mapping of DNA binding sites. eLife 6:e21856.