Cleavage Under Targets and Release Using Nuclease (CUT&RUN)
Geneva Miller, Lindsey M. Rollosson, Carrie Saada, Serenity J. Wade, Danae Schulz
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.
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).
Attachments
Steps
Prepare cells
Count parasite cultures with hemocytometer or other preferred counting method.
Spin down cells in centrifuge at 2800x g.
Remove supernatant and resuspend in small amount of remaining media (~100µL).
If needed, combine samples from multiple Eppendorf tubes so that each final tube has 75 million cells, and spin again at 2800x g,10°C in microcentrifuge. Remove supernatant.
Permeabilize cells
Wash all samples with 1mL NP-S Buffer with 0.1% Saponin.
Spin at 4600x g,10°C. Remove sup.
Primary Antibody Binding
Resuspend each sample in 100µL NP-S Buffer with 0.1% Saponin.
Add EDTA to 2millimolar (mM) final (4µL of 0.05Molarity (M) EDTA/ tube).
Add 5µg α-HA antibody (or other antibody against protein of interest for experimental sample) OR add 5µg α-H3 antibody (for control sample, might require titration) OR add 5µg α-IgG antibody (for control sample).
Incubate for 0h 45m 0s on tube rotator at Room temperature.
Add 1mL NP-S Buffer no detergent to each sample.
Spin at 4600x g,10°C. Remove supernatant.
Add 1mL NP-S Buffer no detergent to each sample.
Take out aliquot of 1 million cells for flow cytometry analysis (~14µL if starting with 75 million cells). Set aside On ice.
Spin the remainder of the sample at 4600x g,10°C. Remove supernatant. (total 2 washes).
pAG-MNase binding
Resuspend each sample in 100µL of buffer NP-S Buffer no detergent.
Add 1.5µL of pAG-MNase enzyme to each sample.
Incubate for 0h 45m 0s at Room temperature on rotator.
FACS Sample preparation
Add 1mL NP-S Buffer no detergent to 1 million cell aliquot prepared above.
Spin at 4600x g.
Resuspend in 100µL NP-S Buffer no detergent.
Stain with α-rabbit IgG PE at 1:200 for 0h 15m 0s at Room temperature.
Wash.
Wash in 1mL NP-S Buffer no detergent (or HMI-9) at 7000rpm. (1/2)
Wash in 1mL NP-S Buffer no detergent (or HMI-9) at 7000rpm.(2/2)
Resuspend in 300µL NP-S Buffer no detergent (or HMI-9).
Transfer sample into flow cytometry tube.
Analyze on flow cytometer.
pAG-MNAse wash
1mL NP-S Buffer no detergent to each sample. Spin at 4600x g,10°C. Remove supernatant.
Add 1mL NP-S Buffer no detergent to each sample.
Spin at 4600x g,10°C. Remove supernatant.
Targeted Digestion Preparation
Make sure to prepare enough 2X Stopbuffer with spike in control.
Targeted Digestion
Resuspend each sample in 100µL buffer NP-S Buffer no detergent.
Incubate at Room temperature for 0h 5m 0s.
Add 2µL 100millimolar (mM) CaCl2 to all samples (final concentration = 2mM), mix by flicking.
Incubate all samples at 25°C for 0h 5m 0s.
Add 100µL 2X Stop buffer to each sample and mix by flicking.
Chromatin Release
Incubate 0h 10m 0s at 37°C to release CUT&RUN fragments from the insoluble nuclear chromatin.
Spin at 4600x g,10°C. Remove supernatant into new tube. SAVE SUPERNATANT.
DNA Extraction
To all samples add 2µL of 10% SDS (final concentration = 0.1%), 3.3µL of 10 proteinase K (165µg/ml), and 1.33µL of 1 RNAse A (6.5µg/ml).
Mix by gentle flicking and incubate for 0h 10m 0s at 70°C.
Purify using Ampure XP beads at 1.8X or phenol chloroform extraction.
Spotlight video
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