TITLE: AutoCUT&RUN: genome-wide profiling of chromatin proteins in a 96 well format on a Biomek
AUTHORS: Derek Janssens, Kami Ahmad, Steven Henikoff

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>CUT&RUN is an antibody-targeted nuclease-cleavage method that profiles the genome-wide occupancy of DNA-binding proteins, histones and chromatin modifying proteins </span><span style = "font-style:italic;">in situ</span><span> with exceptional sensitivity and resolution. Here we provide a protocol to perform CUT&RUN in an automated 96 well format using a Beckman Biomek FX liquid handling robot equipped for magnetic separation and temperature control. To circumvent the need to purify small amounts of DNA prior to library preparation, we developed a method to polish the DNA ends in chromatin fragments for direct ligation of Illumina library adapters. This allows the samples to be kept in a 96 well format throughout the DNA end-polishing and adapter ligation steps. Deproteinated CUT&RUN libraries are purified on the Biomek using Ampure XP magnetic beads both before and after PCR enrichment. This AutoCUT&RUN protocol allows a single operator to generate up to 96 libraries in two days that are ready to be pooled and sequenced. The easy, cost-effective workflow makes AutoCUT&RUN an attractive tool for high-throughput characterization of chromatin features, and because AutoCUT&RUN generates high-quality profiles from frozen tissue samples this method is also suitable for rapidly profiling the epigenetic content of patient samples.</span></div></div>

[STEPS]
?. [Binding cells to beads]
Harvest fresh culture(s) at room temperature and count cells. The AutoCUT&RUN protocol is recommended for 50,000 to 1,000,000 mammalian cells per sample. Tissue samples can also be profiled using AutoCUT&RUN and should be processed either manually or enzymatically into a homogenous suspension of intact cells prior to binding Concanavalin A-coated beads.
SECTION TIMING: 30 min
CRITICAL STEP: All steps prior to the addition of antibody are performed at room temperature (~22 oC) to minimize stress on the cells. Because it is crucial that DNA breakage is minimized throughout the protocol, we recommend that cavitation during resuspension and vigorous vortexing be avoided.
PAUSE POINT: If necessary, cells can be cryopreserved in 10% (vol/vol) DMSO using a Mr. Frosty isopropyl alcohol chamber. Alternatively, cells can simply be washed with PBS, pelleted at 600 X g and then snap frozen following aspiration of all liquid. Similarly, dissected tissue can be snap frozen in a cryogenic vial for processing at a later date.
?. [Binding cells to beads]
Centrifuge 3 min 600 x g at room temperature and withdraw liquid.
?. [Binding cells to beads]
Resuspend in 1.5 mL room temperature Wash Buffer by gently pipetting and transfer if necessary to a 2 mL tube.
?. [Binding cells to beads]
Centrifuge 3 min 600 x g at room temperature and withdraw liquid.
?. [Binding cells to beads]
Repeat steps 3 and 4 two more times.
CRITICAL STEP: Thorough washing removes free sugars and other molecules that can compete for binding to the Concanavalin A coated-beads, ensuring efficient binding and recovery of the cells of interest.
?. [Binding cells to beads]
Resuspend in 1 mL room temperature Wash Buffer by gently pipetting.
?. [Binding cells to beads]
While gently vortexing the cells at room temperature, add the Concanavalin A coated-bead suspension.
?. [Permeabilize cells and bind (primary) antibodies]
Mix well by vigorous inversion to ensure the bead-bound cells are in a homogenous suspension and divide into aliquots in 0.6-mL low-bind tubes, one for each antibody to be used.
SECTION TIMING: 2 hrs–overnight
?. [Binding cells to beads]
Place on tube nutator at room temperature for 5-10 min.
?. [Permeabilize cells and bind (primary) antibodies]
Place on the magnet stand to clear and pull off and discard the liquid.
?. [Permeabilize cells and bind (primary) antibodies]
Place each tube at a low angle on the vortex mixer set to low (~1100 rpm) and squirt 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 beads.
CRITICAL STEP: Permeabilizing the cells with digitonin and chelating divalent cations with EDTA serves to quickly halt metabolic processes and prevent endogenous DNAse activity. This helps to preserve the native chromatin state and reduce background noise in the final CUT&RUN libraries. Thus, it is recommended to work quickly to get cells into Antibody Buffer.
?. [Permeabilize cells and bind (primary) antibodies]
Mix in the primary antibody to a final concentration of 1:100 or to the manufacturer’s recommended concentration for immunofluorescence.
CRITICAL STEP: To evaluate success of the procedure without requiring sequencing, include in parallel a positive control antibody (e.g. anti-H3K27me3) and a negative control antibody (e.g.anti-mouse IgG). Do not include a no-antibody control, as the lack of tethering may allow any unbound pA-MNase to act as a “time-bomb” and digest accessible DNA, resulting in a background of DNA-accessible sites.
?. [Permeabilize cells and bind (primary) antibodies]
Place on the tube nutator at room temperature for 2 hrs.
PAUSE POINT: Antibody incubation may proceed overnight at 4 oC.
? TROUBLESHOOTING:  See Troubleshooting Table in Guidelines
?. [Bind secondary antibody (as required)]
Remove liquid from the cap and side with a quick pulse on a micro-centrifuge.
SECTION TIMING: 1 hr-overnight
CRITICAL STEP: The binding efficiency of Protein A to the primary antibody depends on host species and IgG isotype. For example, Protein A binds well to rabbit and guinea pig IgG but poorly to mouse and goat IgG, and so for these latter antibodies a secondary antibody, such as rabbit anti-mouse is recommended.
CRITICAL STEP: After mixing, but before placing a tube on the magnet stand, a very quick spin on a micro-centrifuge (no more than 100 x g) will minimize carry-over of antibody and pA-MN that could result in overall background cleavages during the digestion step.
?. [Bind secondary antibody (as required)]
Place on the magnet stand to clear (~30 s) and pull off all of the liquid.
?. [Bind secondary antibody (as required)]
Add 150 µL Digitonin Buffer to wash, mix by inversion, or by gentle pipetting using a 1 mL tip if clumps persist, and remove liquid from the cap and side with a quick pulse on a micro-centrifuge.
?. [Bind secondary antibody (as required)]
Repeat Digitonin Buffer wash steps 14-16.
?. [Bind secondary antibody (as required)]
Place on the magnet stand to clear and pull off all of the liquid.
?. [Bind secondary antibody (as required)]
Place each tube at a low angle on the vortex mixer set to low (~1100 rpm) and squirt 150 µL of the Digitonin 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.
?. [Bind secondary antibody (as required)]
Mix in the secondary antibody to a final concentration of 1:100 or to the manufacturer’s recommended concentration.
?. [Bind secondary antibody (as required)]
Place on the tube nutator at 4 oC for ~1 hr or overnight.
?. [Bind Protein A-MNase fusion protein on Biomek]
Set Cooling Unit (filled with antifreeze and routed to Heating/Cooling ALP with Aluminum Cooling Block at position P10) to 0 oC.
SECTION TIMING: ~1.5 hr
CRITICAL STEP: Performing the steps up until this point by hand increases the versatility of the platform, allowing individual users to harvest their cells or tissue of interest and bind any antibody of their choosing. Because the antibody incubation is not time sensitive, samples from multiple users can be synchronized at this step and arrayed on a single plate, allowing the remaining steps to be performed in unison on the Biomek by a single operator.
?. [Bind Protein A-MNase fusion protein on Biomek]
Prepare 4.5 mL of pA-MNase solution per 24 AutoCUT&RUN reactions by mixing the pA-MNase (supplied upon request) to a final concentration of ~700 ng/mL in Digitonin Buffer. Transfer pA-MNase solution into a reservoir and dispense 175 µL into each of the active wells of a labeled V-Bottom Plate using a Multi-Channel Pipette. Place at position P4 on the Biomek deck.
CRITICAL STEP: CUT&RUN is relatively insensitive to the concentration of pA-MNase as is evident from the titration test of two different batches, where increasing the concentration of pA-MNase above ~100 ng/mL resulted in little additional release of H3K27me3-bound nucleosomes from 600,000 human cells after 30 min digestion in a 500 µL volume.
?. [Bind Protein A-MNase fusion protein on Biomek]
Using wide bore 200 µL tips, resuspend Concanavalin A bead-bound cells + Antibodies and array them in a PCR 96 Well Plate. Be sure to record the position of each sample in the plate and stack it on a PCR Plate Rack at position P6 on the Biomek deck.
To prevent the PCR Plate Racks from catching and being carried along with the PCR 96 Well Plate during movement steps, the PCR Plate Racks can be taped down to the stationary ALPs (this issue has not been observed for the ALPAQUA Magnet Stand or the Cold Block).
?. [Bind Protein A-MNase fusion protein on Biomek]
Dispense 1 mL of Digitonin Buffer into each of the active wells of a labeled Deep Well Plate and place it at position P5 on the Biomek deck.
?. [Bind Protein A-MNase fusion protein on Biomek]
Place fresh AP96 200 µL tips at position TL1, the ALPAQUA Magnet Plate at position P3, and an empty labeled Deep Well Plate for collecting liquid waste at position P9 on the Biomek deck.
?. [Bind Protein A-MNase fusion protein on Biomek]
Start the pA-MNase Binding Method.
During the 1 hr mixing step continue on to prepare pA-MNase Reaction Mix and 4X Stop Buffer solutions and keep on Ice for the next step of the reaction.
?. [Targeted chromatin digestion on the Biomek]
Prepare pA-MNase Reaction Mix (50 µL/sample). Keep on Ice until use.
SECTION TIMING: ~1.5 hr
?. [Targeted chromatin digestion on the Biomek]
Prepare 4X Stop Buffer (25 µL/sample). Keep on Ice until use.
CRITICAL STEP: Heterologous spike-in DNA is highly recommended for calibration, as there is too little background cleavage for normalization of samples. Spike-in DNA should be fragmented down to ~200 bp mean size, for example an MNase-treated sample of mononucleosome-sized fragments. As we use the total number of mapped reads as a normalization factor only, very little spike-in DNA is needed. For example, addition of 1.5 pg results in 1,000-10,000 mapped spike-in reads for 1-10 million mapped experimental reads (in inverse proportion).
?. [Targeted chromatin digestion on the Biomek]
When the pA-MNase Binding Method is complete, remove the labeled V-Bottom plate containing residual pA-MNase solution and wash well with DI water (this plate can be reused in subsequent experiments). Then empty the liquid waste from the labeled Deep Well Plate, rinse it well with DI water and again place it at position P9 on the Biomek deck. The recirculating water bath filled with antifreeze should remain at 0 degrees celcius for this method.
?. [Targeted chromatin digestion on the Biomek]
Using a Reservoir and Multi-Channel Pipette dispense 50 µL of pA-MNase Reaction Mix into the active wells of a labeled V-Bottom Plate and place it at position P4 on the Biomek deck. Dispense 25 µL of 4X STOP Buffer in the active wells of a labeled V-Bottom Bottom Plate and place it at position P7 on the Biomek deck. Gently shake the Deep Well Plate containing Digitonin Buffer to ensure digitonin remains suspended in solution and again place it at position P5 on the Biomek deck.
?. [Targeted chromatin digestion on the Biomek]
Place a fresh PCR 96 Well Plate on a PCR Plate Rack at position P8 for accepting digested chromatin. The AP96 200 µL Tips at TL1 can be used again for this method, the ALPAQUA Magnet Plate should remain at position P3, and a free PCR Plate Rack should remain at position P6. The PCR 96 Well Plate containing Concanavalin A bead-bound cells + Antibodies + pA-MNase should remain on the Cooling Block set to 0oC at position P10 on the Biomek deck.
?. [Targeted chromatin digestion on the Biomek]
Ensure the Pause at Step 19 of the pA-MNase Digest Method (after addition of MNase Reaction Mix and before addition of 4X STOP Buffer) is set for the desired digestion time.
CRITICAL STEP: Longer digestion times tend to increase the yield of digested chromatin from each reaction, and can be critical to obtain quality signal from low abundance epitopes (e.g. transcription factors). However, for some antibodies (e.g. anti-H3K27ac) longer digestion times have been observed to increase the amount of non-specific or “background” DNA that is recovered. Because the current platform adds 4X STOP Buffer to all the wells synchronously, setting individual digestion lengths for each reaction is currently not an option, therefore a digestion time of 9-30 min is recommended.
?. [Targeted chromatin digestion on the Biomek]
Start the pA-MNase Digest Method.
During the chromatin release step, when the plate is mixing and incubating at room temperature for about half an hour, prepare 4X End Repair and A-Tailing Buffer and keep on Ice for the next step of the reaction.
?. [Chromatin end repair and dA-tailing]
Prepare 4X End Repair and A-tailing (ERA) buffer (12.5 µL/sample). Keep on Ice until use.
SECTION TIMING: ~2.5 hr
?. [Chromatin end repair and dA-tailing]
When the pA-MNase Digestion Method is complete, remove the labeled V-Bottom plates containing residual pA-MNase Reaction Mix and 4X Stop Buffer, as well as the labeled Deep Well Plates containing residual Digitonin Buffer and Liquid Waste from the Biomek deck. Empty the plates and wash well with DI water (these plates can be reused in subsequent experiments). Seal the PCR 96 Well Plate containing the digested Con-A bead-bound cells (position P3) with an adhesive cover and store at -20 oC for potential troubleshooting. Turn off the recirculating water bath.
?. [Chromatin end repair and dA-tailing]
Remove the PCR 96 Well Plate containing digested chromatin (position P8) from the Biomek deck. Using a Reservoir and Multi-Channel Pipette add 12.5 µL 4X ERA buffer to each sample and mix by pipetting up and down 5 times.
?. [Chromatin end repair and dA-tailing]
Seal the PCR 96 Well Plate using an adhesive cover and place in a thermocycler that has been pre-cooled to 12oC and run the following program with heated lid for temp >20oC:
?. [Adapter Ligation]
Remove pre-annealed 0.15 µM TruSeq adapters from freezer and allow to thaw on ice.
SECTION TIMING: 1 hr-overnight
CRITICAL STEP: To facilitate multiplexing during sequencing each adapter includes a six-nucleotide index or “barcode”. For subsequent data analysis make sure to keep track of which adapter is used for each sample. To allow for addition of adapters using a Multi-Channel Pipette 0.15 µM pre-annealed TruSeq adapters can be arrayed in a 96 Well PCR plate.
?. [Adapter Ligation]
Prepare 2X Rapid Ligase solution (50 µL/ sample). Keep on Ice until use.
?. [Adapter Ligation]
Using a Multi-Channel Pipette, add 5 µL of 0.15 µM pre-annealed TruSeq adapters to each sample and mix by pipetting up and down 5 times.
?. [Adapter Ligation]
Using a Reservoir and Multi-Channel Pipette, add 50 µL 2X Rapid Ligase solution to each sample and mix well by pipetting up and down 10 times.
?. [Adapter Ligation]
Seal the PCR 96 Well Plate using an adhesive cover and place in a thermocycler that has been pre-cooled to 20oC and incubate at 20oC for 20 min. Following incubation proceed with Proteinase K digestion immediately.
?. [Adapter Ligation]
Using two Reservoirs and a Multi-Channel Pipette, add 2 µL 10% SDS and 2 µL Proteinase K (20 mg/mL) to each sample. Seal the PCR 96 Well Plate using an adhesive cover and mix well by vortexing. Spin down solution @ 2,000 rpm for 1 min and incubate in a thermocycler at 37oC for 30 min-overnight.
?. [Pre-PCR DNA Cleanup on the Biomek]
Remove Ampure Bead Slurry from refrigerator, resuspend beads by vortexing and allow to equilibrate to room temperature before using.
SECTION TIMING: ~2 hrs
CRITICAL STEP: Two rounds of Ampure Bead Cleanup are performed prior to PCR amplification to remove un-ligated, and self-ligated adapter as well as unwanted protein, PEG, and salt.
?. [Pre-PCR DNA Cleanup on the Biomek]
Spin PCR 96 Well Plate containing Adapter Ligated DNA samples @ 2,000 rpm for 1 min. Remove seal and stack on a PCR Plate Rack positioned at P5 on the Biomek deck.
?. [Pre-PCR DNA Cleanup on the Biomek]
Using a Reservoir and Multi-Channel Pipette distribute 90 µL of the Ampure Bead Slurry into the active wells of a PCR 96 Well Plate and stack on a PCR Plate Rack positioned at P6 on the Biomek deck.
?. [Pre-PCR DNA Cleanup on the Biomek]
Dispense 1 mL of 80% Ethanol into each of the active wells of a labeled Deep Well Plate and place at position P4 on the Biomek deck.
?. [Pre-PCR DNA Cleanup on the Biomek]
For up to 24 AutoCUT&RUN reactions prepare 6 mL of HXP Mix (20% PEG 8000, 2.5M NaCl). Using a Reservoir and Multi-Channel Pipette distribute 100 µL of the HXP Mix into the active wells of a V-Bottom Plate and place at position P7 on the Biomek deck.
CRITICAL STEP: HXP Mix is light sensitive and will degrade over time once mixed. Therefore, the HXP Mix should either be prepared fresh for each use or stored in the dark at -20oC.
?. [Pre-PCR DNA Cleanup on the Biomek]
Using a Reservoir and Multi-Channel Pipette distribute 100 µL of 10mM Tris-HCl pH 8 into the active wells of two labeled V-Bottom Plates (one for tip washes and one for DNA elution) and place at positions P1 and P2 on the Biomek deck.
?. [Pre-PCR DNA Cleanup on the Biomek]
Place Fresh AP96 200 µL Tips at TL1, a labeled Deep Well Plate for accepting liquid waste at position P9, and a fresh PCR 96 Well Plate stacked on a PCR Plate Rack for accepting clean Adapter Ligated DNA in position P8 on the Biomek deck.
?. [Pre-PCR DNA Cleanup on the Biomek]
Start the Pre-PCR DNA Cleanup Method.
After about 1hr continue on to prepare KAPA PCR Master Mix and keep on Ice for the next step of the reaction.
?. [PCR Amplification of CUT&RUN Libraries.]
Prepare KAPA PCR Master Mix.
SECTION TIMING: 1 hr-overnight
?. [PCR Amplification of CUT&RUN Libraries.]
When the Pre-PCR DNA Cleanup Method is complete, remove the labeled V-Bottom Plates containing residual 10mM Tris-HCl pH 8 and HXP Mix, as well as the labeled Deep Well Plates containing residual Ethanol and Liquid Waste from the Biomek deck. Empty the plates and wash well with DI water (these plates can be reused in subsequent experiments).
?. [PCR Amplification of CUT&RUN Libraries.]
Remove PCR 96 Well Plate containing clean Adapter Ligated DNA (position P8) from the Biomek deck. Using a Reservoir and Multi-Channel Pipette add 20 µL KAPA PCR Master Mix to each sample and mix well by pipetting up and down 10 times.
?. [PCR Amplification of CUT&RUN Libraries.]
Seal the PCR plate using an adhesive cover, place in thermocycler and run the following program with heated lid:
CRITICAL STEP: Libraries should be amplified with 12-14 PCR cycles, preferably with a 10 s 60oC combined annealing/extension step to minimize the contribution of large DNA fragments to the library. Good results have been obtained with the Hyper-prep kit (KAPA Biosystems).
?. [Post-PCR DNA Cleanup on the Biomek]
Remove Ampure Bead Slurry from refrigerator, resuspend beads by vortexing and allow to equilibrate to room temperature before using.
SECTION TIMING: ~2 hr
CRITICAL STEP: Two rounds of Ampure Bead Cleanup are performed following PCR amplification to remove unused P5 and P7 primers, amplified self-ligated adapters and unwanted protein and salt. Once amplified, the CUT&RUN library can serve as template for subsequent PCR reactions using the P5 and P7 primers, making cross-contamination of Pre-PCR samples a serious concern. Therefore, all Post-PCR reagents should be stored separately from Pre-PCR reagents, and prepared in a separate area. If labware is to be reused, separate plates should be clearly labeled for use in Post-PCR reactions only.
?. [Post-PCR DNA Cleanup on the Biomek]
Spin PCR 96 Well Plate containing PCR amplified CUT&RUN libraries @2,000 rpm for 1 min. Remove seal and stack on a PCR Plate Rack positioned at P5 on the Biomeck deck.
?. [Post-PCR DNA Cleanup on the Biomek]
Using a Reservoir and Multi-Channel Pipette distribute 55 µL of the Ampure Bead Slurry into the active wells of a PCR 96 Well Plate and stack on a PCR Plate Rack positioned at P6 on the Biomek deck.
?. [Post-PCR DNA Cleanup on the Biomek]
Dispense 1 mL of 80% Ethanol into each of the active wells of a labeled Deep Well Plate and place at position P4 on the Biomek Deck.
?. [Post-PCR DNA Cleanup on the Biomek]
Using a Reservoir and Multi-Channel Pipette distribute 100 µL of the remaining HXP Mix into the active wells of a V-Bottom Plate and place at position P7 on the Biomek deck.
?. [Post-PCR DNA Cleanup on the Biomek]
Using a Reservoir and Multi-Channel Pipette distribute 100 µL of 10mM Tris-HCl pH 8 into the active wells of two V-Bottom Plates (one for tip washes and one for DNA elution) and place at positions P1 and P2 on the Biomek deck.
?. [Post-PCR DNA Cleanup on the Biomek]
Place Fresh AP96 200 µL Tips at TL1, a labeled Deep Well Plate for accepting liquid waste at position P9, and a fresh PCR 96 Well Plate stacked on a PCR Plate Rack for accepting clean CUT&RUN DNA Libraries at position P8 on the Biomek deck.
?. [Post-PCR DNA Cleanup on the Biomek]
Start the Post-PCR DNA Cleanup Method.
?. [Sequencing]
When the Post-PCR DNA Cleanup Method is complete, remove the labeled V-Bottom plates containing residual 10mM Tris-HCl pH 8 and HXP Buffer, as well as the labeled Deep Well Plates containing residual Ethanol and Liquid Waste from the Biomek deck. Empty the plates and wash extremely well with DI water (these plates can be reused in subsequent experiments).
SECTION TIMING: 1-2 days
?. [Sequencing]
Remove PCR 96 Well Plate containing clean CUT&RUN Libraries (position P8) from the Biomek deck and seal with an adhesive cover for subsequent analysis.
?. [Sequencing]
Determine the size distribution of libraries by Agilent 4200 TapeStation analysis.
? TROUBLESHOOTING:  See Troubleshooting Table in Guidelines
?. [Sequencing]
Quantify library yield using dsDNA-specific assay, such as Qubit.
?. [Sequencing]
Pool indexed CUT&RUN libraries and perform paired-end Illumina sequencing following the manufacturer’s instructions.
CRITICAL STEP: Because of the very low background with CUT&RUN, typically 5 million paired-end reads suffices for transcription factors or nucleosome modifications, even for the human genome. For maximum economy, we mix up to 24 barcoded samples per lane at equimolar concentration (proved a similar number of reads is desired for each sample) and perform paired-end 25x25 bp sequencing on a 2-lane flow cell. Single-end sequencing is not recommended for CUT&RUN, as it sacrifices resolution and discrimination between transcription factors and neighboring nucleosomes.
?. [Data processing and analysis]
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 TIMING: (variable)
CRITICAL STEP: Separation of sequenced fragments into ≤120 bp and ≥150 bp size classes provides mapping of the local vicinity of a DNA-binding protein, but this can vary depending on the steric access to the DNA by the tethered MNase.
? TROUBLESHOOTING:  See Troubleshooting Table in Guidelines