CUT&RUN abbreviated protocol

We previously described a novel alternative to Chromatin Immunoprecipitation, Cleavage Under Targets & Release Using Nuclease (CUT&RUN), in which unfixed permeabilized cells are incubated with antibody, followed by binding of a Protein A-Micrococcal Nuclease (pA/MNase) fusion protein. Upon activation of tethered MNase, the bound complex is excised and released into the supernatant for DNA extraction and sequencing. In the manuscript attached to this version of the protocol, Meers et al. introduce four enhancements to CUT&RUN: 1) a hybrid Protein A-Protein G-MNase construct that expands antibody compatibility; 2) a modified digestion protocol that prevents premature release of the nuclease-bound complex; 3) a calibration strategy based on carry-over of E. coli DNA introduced with the fusion protein; and 4) a novel peak-calling strategy customized for the low-background profiles obtained using CUT&RUN. Here we provide an updated CUT&RUN protocol that incorporates these enhancements, and provides three different options for the CUT&RUN MNase digestion reaction that are helpful to improve data quality or to increase throughput. These new features, coupled with the previously described low-cost, high efficiency, high reproducibility and high-throughput capability of CUT&RUN make it the method of choice for routine epigenomic profiling.

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.

Protocol Option 1: Standard CUT&RUN specific reagents

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

• CRITICAL STEP : Adding heterologous spike-in DNA to the STOP Buffer can be useful for comparison of DNA yields between samples. The total number of mapped spike-in reads can then be used as a normalization factor, where the amount of spike-in reads is inversly proportional to the DNA yeild from the sample. The spike-in DNA should be fragmented down to ~200 bp mean size, for example, an MNase-treated sample of mononucleosome-sized fragments. When starting with low cell numbers (i.e. 100 - 10,000 cells) very little spike-in DNA is required, we recommend a final concentration of 2 pg/mL in the STOP buffer. For samples with high cell numbers (i.e. 10,000 -1 million cells) more spike-in DNA is required to obtain sufficient reads and we recommend 100 pg/mL in the STOP Buffer. Alternatively, E. coli DNA that is carried-over from the production of the fusion protein can also serve as a spike-in for sample calibrations, in which case no additional heterologous spike-in needs to be included in the STOP Buffer.

Protocol Option 2: High Ca2+ / Low Salt specific reagents ²⁺ / Low Salt specific reagents

Low-Salt Rinse Buffer: Mix 20 mL of Low-Salt Rinse Buffer. Store the buffer at 4 °C for up to 1 week.

Incubation Buffer: Mix 4 mL of Incubation Buffer. Store the buffer at 4 °C for up to 1 week. Briefly chill on ice before use.

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

CRITICAL STEP : Adding heterologous spike-in DNA to the STOP Buffer can be useful for comparison of DNA yields between samples. For recommended concentrations see 2X STOP Buffer in Protocol Option 1 specific reagents. Alternatively, E. coli DNA that is carried-over from the production of the fusion protein can also serve as a Spike-In for sample calibrations, in which case no additional heterologous spike-in needs to be included in the STOP Buffer.

Protocol Option 3: Direct Ligation specific reagents

1X pA-MNase Reaction Mix: Prepare 1.2 mL of 1X pA-MNase Reaction Mix. Store the buffer at 4 °C for up to 1 week.

4X STOP Buffer: Mix 600 µL of 4X STOP Buffer. Store the buffer at 4 °C for up to 1 week.

CRITICAL STEP : Adding heterologous spike-in DNA to the STOP Buffer can be useful for comparison of DNA yields between samples. For recommended concentrations see 2X STOP Buffer in Protocol Option 1 specific reagents. Alternatively, E. coli DNA that is carried-over from the production of the fusion protein can also serve as a Spike-In for sample calibrations, in which case no additional heterologous spike-in needs to be included in the STOP Buffer.