TITLE: Cleavage Under Targets and Release Using Nuclease (CUT&amp;RUN)
AUTHORS: Geneva Miller, Lindsey M. Rollosson, Carrie Saada, Serenity J. Wade, Danae Schulz

[DESCRIPTION]
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. 

[BEFORE_START]
Before Start

We use 50-75 million bloodstream parasites per sample. Parasites are cultured in HMI9 media with incubation at 37 °Cand 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).


[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.



[STEPS]
SECTION: Prepare cells
1. Count parasite cultures with hemocytometer or other preferred counting method.
SECTION: Prepare cells
2. Spin down cells in centrifuge at 2800 x g, 10 min. 
 

SECTION: Prepare cells
3. If needed, combine samples from multiple Eppendorf tubes so that each final tube has 75 million cells, and spin again at 2800 x g, 4 min, 10 °C in microcentrifuge. Remove supernatant.

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

SECTION: Permeabilize cells
5. Spin at 4600 x g, 4 min, 10 °C. Remove sup.
SECTION: Primary Antibody Binding
6. Resuspend each sample in 100 µL NP-S Buffer with 0.1% Saponin.

SECTION: Primary Antibody Binding
7. Add EDTA to 2 millimolar (mM) final (4 µL of 0.05 Molarity (M) EDTA/ tube).
SECTION: Primary Antibody Binding
8. 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).
SECTION: Primary Antibody Binding
9. Incubate for 45 min on tube rotator at Room temperature.

SECTION: Primary Antibody Binding
10. Add 1 mL NP-S Buffer no detergent to each sample.
SECTION: Primary Antibody Binding
11. Spin at 4600 x g, 4 min, 10 °C. Remove supernatant.

SECTION: Primary Antibody Binding
12. Add 1 mL NP-S Buffer no detergent to each sample.

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

SECTION: pAG-MNase binding
16. Add 1.5 µL of pAG-MNase enzyme to each sample.
SECTION: pAG-MNase binding
17. Incubate for 45 min at Room temperature on rotator.

SECTION: FACS Sample preparation
18. Add 1 mL NP-S Buffer no detergent to 1 million cell aliquot prepared above.
 

SECTION: FACS Sample preparation
19. Spin at 4600 x g, 4 min.

SECTION: FACS Sample preparation
20. Resuspend in 100 µL NP-S Buffer no detergent.
SECTION: FACS Sample preparation
21. Stain with α-rabbit IgG PE at 1:200 for 15 min at Room temperature.
SECTION: FACS Sample preparation
22. Wash.
SECTION: FACS Sample preparation
23. Resuspend in 300 µL NP-S Buffer no detergent (or HMI-9).
SECTION: FACS Sample preparation
24. Transfer sample into flow cytometry tube.
SECTION: FACS Sample preparation
25. Analyze on flow cytometer.
SECTION: pAG-MNAse wash
26. 
 
Add 1 mL NP-S Buffer no detergent to each sample.
SECTION: pAG-MNAse wash
27. Spin at 4600 x g, 4 min, 10 °C. Remove supernatant.

SECTION: pAG-MNAse wash
28. Add 1 mL NP-S Buffer no detergent to each sample.
SECTION: pAG-MNAse wash
29. Spin at 4600 x g, 4 min, 10 °C. Remove supernatant.
SECTION: Targeted Digestion Preparation
30. Make sure to prepare enough 2X Stopbuffer with spike in control.
 

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

SECTION: Targeted Digestion
32. Incubate at Room temperature for 5 min.
SECTION: Targeted Digestion
33. Add 2 µL 100 millimolar (mM) CaCl2 to all samples (final concentration = 2mM), mix by flicking.
SECTION: Targeted Digestion
34. Incubate all samples at 25 °C for 5 min. 
 


SECTION: Targeted Digestion
35. Add 100 µL 2X Stop buffer to each sample and mix by flicking.
SECTION: Chromatin Release
36. Incubate 10 min at 37 °C to release CUT&RUN fragments from the insoluble nuclear chromatin.
SECTION: Chromatin Release
37. Spin at 4600 x g, 4 min, 10 °C. Remove supernatant into new tube. SAVE SUPERNATANT.

SECTION: DNA Extraction
38. To all samples add 2 µL of 10% SDS (final concentration = 0.1%), 3.3 µL of 10 1798 proteinase K (165µg/ml), and 1.33 µL of 1 1798 RNAse A (6.5µg/ml).
SECTION: DNA Extraction
39. Mix by gentle flicking and incubate for 10 min at 70 °C.

SECTION: DNA Extraction
40. Purify using Ampure XP beads at 1.8X or phenol chloroform extraction.
 

SECTION: Prepare cells
2.1. Remove supernatant and resuspend in small amount of remaining media (~100 µL).
SECTION: FACS Sample preparation
22.1. Wash in 1 mL NP-S Buffer no detergent (or HMI-9) at 7000 rpm, 4 min. (1/2)

SECTION: FACS Sample preparation
22.2. Wash in 1 mL NP-S Buffer no detergent (or HMI-9) at 7000 rpm, 4 min.(2/2)