TITLE: CUT&RUN: Targeted in situ genome-wide profiling with high efficiency for low cell numbers
AUTHORS: Derek Janssens, Steven Henikoff

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>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 </span><span style = "font-style:italic;">E. coli </span><span>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.</span></div></div>

[STEPS]
?. [Binding cells to beads (~30 min)]
Harvest fresh culture(s) at room temperature and count cells. The same protocol can be used for up to 500,000 mammalian cells per sample and/or digestion time point.
CRITICAL STEP: All steps prior to the addition of antibody are performed at room temperature 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% DMSO using a Mr. Frosty isopropyl alcohol chamber. We do not recommend flash freezing, as this can cause background DNA breakage that may impact final data quality.
?. [Binding cells to beads (~30 min)]
Centrifuge 3 min 600 x g at room temperature and withdraw liquid.
[Centrifugation]
?. [Binding cells to beads (~30 min)]
Resuspend in 1.5 mL room temperature Wash buffer by gently pipetting and transfer if necessary to a 2 mL tube.
[Wash buffer]
?. [Permeabilize cells and bind primary antibodies (2.5 hours - overnight)]
Place on the magnet stand to clear and pull off the liquid.
?. [Permeabilize cells and bind primary antibodies (2.5 hours - overnight)]
Place each tube at a low angle on the vortex mixer set to low (~1100 rpm) and squirt 50-150 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
[Antibody buffer]
NOTE: 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.
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. rabbit 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 (2.5 hours - overnight)]
Place on a nutator or tube rotator at room temperature for ~2 hr or at 4 °C overnight.
[Nutator or tube rotator at RT]
PAUSE POINT: Antibody incubation may proceed overnight at 4 °C.
NOTE: When using on the order of 500K cells per 10 µL beads it is normal for beads to clump or stick to the side during the incubation. First, attempt to resuspend stuck beads by inverting the tube. If beads remain stuck to the side, use a pipette tip to push beads back into solution.
TROUBLESHOOTING
?. [Bind secondary antibody as required (15 min to 1.5 hours)]
Remove liquid from the cap and side with a quick pulse on a micro-centrifuge. Place on the magnet stand to clear (~30 s) and pull off all of the liquid.
[Magnet stand]
?. [Bind secondary antibody as required (15 min to 1.5 hours)]
Place each tube at a low angle on the vortex mixer set to low (~1100 rpm) and squirt 150 μL of the Dig-wash 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.
[Dig-Wash Buffer (per sample or digestion time point)]
?. [Bind secondary antibody as required (15 min to 1.5 hours)]
Place on the nutator or tube rotator at 4 °C for ~1 hr.
[Nutator or tube rotator at 4 °C]
?. [Bind Protein A-MNase or Protein A/G-MNase fusion protein (1.5 hours)]
Place each tube at a low angle on the vortex mixer set to low (~1100 rpm). Squirt 150 μL of the Protein A-MNase or Protein A/G-MNase fusion protein at 700 ng/mL (e.g., 1:200 of a 140 µg/mL glycerol stock) in Dig-wash 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.
[Protein A-MNase  or Protein A/G-MNase (700 ng/mL) in Dig-wash buffer]
?. [Bind Protein A-MNase or Protein A/G-MNase fusion protein (1.5 hours)]
Place on the nutator or tube rotator at 4 °C for ~1 hr.
[Nutator or tube rotator at 4 °C]
?. [Chromatin Digestion and Release Option 2: High Ca2+ / Low Salt (1 hour)]
Remove liquid from the cap and side with a quick pulse on a micro-centrifuge. Place on the magnet stand to clear and pull off all of the liquid. Add 1 mL Low-Salt Rinse Buffer, mix by inversion, or by gentle pipetting if clumps persist, and remove liquid from cap and side with a quick pulse on a micro-centrifuge.
[Low-Salt Rinse buffer]
CRITICAL STEP: If multiple digestion time points are desired per sample, split samples into multiple tubes for MNase digestion and STOP steps, one tube for each time point.
?. [Phenol Chloroform Extraction (~1.5 hours)]
To each sample add 2 μL 10% SDS (to 0.1%), and 2.5 μL Proteinase K (20 mg/ml). Mix by inversion and incubate 1 hr @ 50 °C.
[10% SDS (to 0.1%)/sample]
[Proteinase K (20 mg/ml)/sample]
[Incubation]
[Incubation]
NOTE: If you are running protocol option 3 (Direct Ligation) skip this section.
?. [Phenol Chloroform Extraction (~1.5 hours)]
Add an equal volume of Phenol Chloroform to the sample (e.g. to 200 µL sample add 200 µL Phenol Chloroform). Mix by full-speed vortexing ~2 s.
[PCl]
[Vortexing]
?. [Phenol Chloroform Extraction (~1.5 hours)]
Transfer to a phase-lock tube (e.g., Qiagen MaXtract), and centrifuge 5 min, room temperature @ 16,000 x g.
[Centrifugation]
?. [Phenol Chloroform Extraction (~1.5 hours)]
Add an equivalent volume of chloroform to the initial sample volume (e.g. for a 200 µL starting sample volume add 200 µL chloroform). Invert ~10X to mix and then centrifuge 5 min, room temperature @ 16,000 x g.
[Chloroform]
[Centrifugation]
?. [Phenol Chloroform Extraction (~1.5 hours)]
Remove the top liquid phase by pipetting to a fresh tube containing 2 μL 2 mg/mL glycogen (diluted 1:10 from 20 mg/mL glycogen stock).
[2 mg/ml glycogen]
?. [Phenol Chloroform Extraction (~1.5 hours)]
Add 500 μL 100% ethanol and mix by vortexing or tube inversion.
[100% ethanol]
?. [Phenol Chloroform Extraction (~1.5 hours)]
Chill on ice and centrifuge 10 min, 4 °C @ 16,000 x g.
[Centrifugation]
[Centrifugation]
?. [Phenol Chloroform Extraction (~1.5 hours)]
Pour off the liquid and drain on a paper towel.
?. [Phenol Chloroform Extraction (~1.5 hours)]
Rinse the pellet in 1 ml 100% ethanol and centrifuge 1 min, 4 °C @ 16,000 x g.
[100% ethanol]
[Centrifugation]
[Centrifugation]
?. [Phenol Chloroform Extraction (~1.5 hours)]
Carefully pour off the liquid and drain on a paper towel. Air dry.
[Air Dry]
?. [Phenol Chloroform Extraction (~1.5 hours)]
When the pellet is dry, dissolve in 30-50 μL 1 mM Tris-HCl pH 8 0.1 mM EDTA, then transfer to a new 0.6 mL Lo-Bind microcentrifuge tube.
[1 mM Tris-HCl pH 8 0.1 mM EDTA]
?. [Sample Analysis Pre-Library Prep (~1 hour)]
Optional: Quantify 1-2 μL, for example using fluorescence detection with a Qubit instrument.
NOTE: If running protocol option 3 (Direct Ligation) skip this step as the STOP buffer is not a suitable solution for either Qubit or TapeStation analysis.
TROUBLESHOOTING
?. [Sample Analysis Pre-Library Prep (~1 hour)]
Optional: Evaluate the presence of cleaved fragments and the size distribution by capillary electrophoresis with fluorescence detection, for example using a Tapestation instrument.
CRITICAL STEP: Some long undigested DNA will leak through, and this is what will dominate the Qubit fluorescence for CUT&RUN of typical transcription factors. For these, the targeted DNA recovered is too low in amount and too small in size to be detected by gel analysis or even by Tapestation. In such cases it may be necessary to make a PCR-amplified library to quantify by Tapestation or Bioanalyzer analysis.
TROUBLESHOOTING
?. [CUT&RUN Library Analysis and Sequencing]
Quantify library yield using dsDNA-specific assay, such as Qubit.
TROUBLESHOOTING
?. [CUT&RUN Library Analysis and Sequencing]
Determine the size distribution of libraries by Agilent 4200 TapeStation analysis.
TROUBLESHOOTING
?. [CUT&RUN Library Analysis and Sequencing]
Pool samples at equimolar concentrations and perform paired-end Illumina sequencing on the barcoded libraries 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 48 barcoded samples and sequence the pool on an entire 2-lane flow cell, and perform paired-end 25x25 bp sequencing. 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.
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. Single-end sequencing is not recommended for CUT&RUN, as it sacrifices resolution and discrimination between transcription factors and neighboring nucleosomes.
TROUBLESHOOTING
?. [Data Processing and Analysis]
Scripts are available from https://github.com/Henikoff/Cut-and-Run for spike-in calibration and for peak-calling.
TROUBLESHOOTING
?. [Binding cells to beads (~30 min)]
Centrifuge 3 min 600 x g at room temperature and withdraw liquid.
[Centrifugation]
?. [Binding cells to beads (~30 min)]
While gently vortexing the cells at room temperature, add the ConA-coated magnetic bead slurry.
?. [Binding cells to beads (~30 min)]
Rotate 5-10 min at room temperature.
[Rotation]
?. [Binding cells to beads (~30 min)]
Repeat steps 3 and 4 two more times.
[Repeat Dig-wash steps]
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 (~30 min)]
Resuspend in 1 mL room temperature Wash Buffer by gently pipetting.
?. [Permeabilize cells and bind primary antibodies (2.5 hours - overnight)]
Mix well by vigorous inversion to ensure the bead-bound cells are in a homogenous suspension and divide into aliquots in 1.5-mL tubes, one for each antibody to be used.
NOTE: Some users have experienced issues with ConA beads sticking to the sides of the tube and coming out of solution during antibody incubation steps. In this case, 0.6-mL Lo-bind microcentrifuge tubes can be used, and subsequent Dig-wash volumes reduced to 300 µL. However, when bound to low cell numbers (e.g.
?. [Permeabilize cells and bind primary antibodies (2.5 hours - overnight)]
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. rabbit 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.
?. [Bind secondary antibody as required (15 min to 1.5 hours)]
Add 1 mL Dig-wash buffer, mix by inversion, or by gentle pipetting using a 1 mL tip if clumps persist.
[Dig-wash buffer]
?. [Bind secondary antibody as required (15 min to 1.5 hours)]
Remove liquid from the cap and side with a quick pulse on a micro-centrifuge. Place on the magnet stand to clear (~30 s) and pull off all of the liquid.
[Magnet stand]
CRITICAL STEP: The binding efficiency of Protein A and Protein A/G 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 α-mouse is recommended. Although Protein A/G increases antibody compatibility relative to Protein A, in some cases a secondary antibody may still be necessary.
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 (15 min to 1.5 hours)]
Repeat Dig-wash steps 14-15.
[Repeat Dig-wash steps]
?. [Bind Protein A-MNase or Protein A/G-MNase fusion protein (1.5 hours)]
Remove liquid from the cap and side with a quick pulse on a micro-centrifuge. Place on the magnet stand to clear (~30 s) and pull off all of the liquid.
[Magnet stand]
?. [Bind Protein A-MNase or Protein A/G-MNase fusion protein (1.5 hours)]
Remove liquid from the cap and side with a quick pulse on a micro-centrifuge. Place on the magnet stand (~30 sec) to clear and pull off all of the liquid.
[Magnet stand]
?. [Bind Protein A-MNase or Protein A/G-MNase fusion protein (1.5 hours)]
Add 1 mL Dig-Wash buffer, mix by inversion, or by gentle pipetting if clumps persist.
[Dig-Wash buffer]
?. [Bind Protein A-MNase or Protein A/G-MNase fusion protein (1.5 hours)]
Repeat Dig-wash steps 21-22.
[Repeat Dig-wash steps]
?. [Chromatin Digestion and Release Option 2: High Ca2+ / Low Salt (1 hour)]
Repeat Dig-wash steps 38-39.
[Repeat Dig-wash steps]
?. [Chromatin Digestion and Release Option 2: High Ca2+ / Low Salt (1 hour)]
Add 1 mL Dig-wash buffer, mix by inversion, or by gentle pipetting if clumps persist.
[Dig-wash buffer]
?. [Chromatin Digestion and Release Option 2: High Ca2+ / Low Salt (1 hour)]
Remove liquid from the cap and side with a quick pulse on a micro-centrifuge. Place on the magnet stand (~30 sec) to clear and pull off all of the liquid.
NOTE: The modified High Ca2+ / Low Salt CUT&RUN digestion protocol prevents premature release of the nuclease-bound complex, thereby preventing premature diffusion of the pA- or pA/G-MNase complex which has the potential to cause non-specific cleavage and increase the background signal. This option is ideal for targets that are prevalent at regions of active chromatin, as well as optimizing conditions for an antibody that appears to yield some specific signal using the standard CUT&RUN protocol but has unacceptably high levels of background signal.
[Magnet stand]
?. [Chromatin Digestion and Release Option 1: Standard CUT&RUN (1.5 hours)]
Place each tube at a low angle on the vortex mixer set to low (~1100 rpm) and add 100 μL of the Dig-wash 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.
[Dig-wash buffer (per sample or digestion time point)]
?. [Chromatin Digestion and Release Option 1: Standard CUT&RUN (1.5 hours)]
Remove liquid from the cap and side with a quick pulse on a micro-centrifuge. Place on the magnet stand (~30 s) to clear and pull off all of the liquid.
NOTE: The Standard CUT&RUN protocol is suitable for most target proteins and is typically the best option to start with for untested antibodies.
[Magnet stand]
?. [Chromatin Digestion and Release Option 1: Standard CUT&RUN (1.5 hours)]
Add 1 mL Dig-wash buffer, mix by inversion, or by gentle pipetting if clumps persist.
[Dig-wash buffer]
?. [Chromatin Digestion and Release Option 1: Standard CUT&RUN (1.5 hours)]
Repeat Dig-wash steps 27-28.
[Repeat Dig-wash steps]
?. [Chromatin Digestion and Release Option 1: Standard CUT&RUN (1.5 hours)]
Insert tubes into the 1.5 mL wells of a heater block sitting in wet ice to chill down to 0 °C.
?. [Chromatin Digestion and Release Option 1: Standard CUT&RUN (1.5 hours)]
Remove each tube from the block, mix in 2 μL 100 mM CaCl2 (per sample and/or digestion time point), diluted 1:10 from a 1 M stock, with gentle vortexing and immediately replace the tube in the 0 °C block
[100 mM CaCl2 (per sample or digestion time point)]
?. [Chromatin Digestion and Release Option 1: Standard CUT&RUN (1.5 hours)]
Incubate at 0 °C for the desired digestion time (default is 30 min).
[Incubation]
[Incubation]
CRITICAL STEP: MNase binds DNA but only cleaves when Ca++ is present, so that digestion is a zero-order reaction that seems to be less temperature-dependent than the subsequent diffusion of released pA-MNase-bound particles that can digest accessible regions of the genome. Cleavage and release of particles in most of the cell population can be obtained at 0 °C while minimizing background cleavages attributable to diffusion. We have found that digestion at ambient temperature or higher results in unacceptable background cleavage levels.
?. [Chromatin Digestion and Release Option 1: Standard CUT&RUN (1.5 hours)]
Add 100 μL 2X STOP Buffer and mix by gentle vortexing. When there are multiple digestion time points, remove 100 μL and add to a new tube containing 100 μL 2X STOP Buffer and mix by gentle vortexing.
[2XSTOP]
CRITICAL STEP: Heterologous spike-in DNA should be present in the 2X STOP Buffer to calibrate DNA amounts, for example to compare treatments or digestion time points. This is especially important for CUT&RUN, as there is too little background cleavage for normalization of samples. Alternatively, mapping to E. coli carry-over DNA from the pA-MNase and pA/G-MNase that gets fragmented during digestion suffices for calibration.
?. [Chromatin Digestion and Release Option 1: Standard CUT&RUN (1.5 hours)]
Incubate 30 min @ 37 °C to release CUT&RUN fragments from the insoluble nuclear chromatin.
[Incubation]
[Incubation]
?. [Chromatin Digestion and Release Option 2: High Ca2+ / Low Salt (1 hour)]
Place on the magnet stand to clear (~30 s) and pull off all of the liquid.
[Magnet stand]
?. [Chromatin Digestion and Release Option 2: High Ca2+ / Low Salt (1 hour)]
Place each tube at a low angle on the vortex mixer set to low (~1100 rpm) and add 200 μL of ice cold Incubation Buffer along the side while gently vortexing to allow the solution to dislodge most or all of the beads. Tap to dislodge the remaining beads.
[Incubation buffer]
?. [Chromatin Digestion and Release Option 2: High Ca2+ / Low Salt (1 hour)]
Incubate at 0 °C for the desired digestion time (default is 5 min).
[Incubation]
CRITICAL STEP: MNase binds DNA but only cleaves when Ca++ is present, so that digestion is a zero-order reaction that seems to be less temperature-dependent than the subsequent diffusion of released pA-MNase-bound particles that can digest accessible regions of the genome. Cleavage and release of particles in most of the cell population can be obtained at 0 °C while minimizing background cleavages attributable to diffusion. We have found that digestion at ambient temperature or higher results in unacceptable background cleavage levels.
[Incubation]
?. [Chromatin Digestion and Release Option 2: High Ca2+ / Low Salt (1 hour)]
Place on (cold) magnet stand and allow to clear for ≥10 s and remove liquid. Add 200 μL STOP buffer and mix by gentle vortexing.
[STOP]
NOTE: The 10 mM Ca++ in the Incubation Buffer will  compact chromatin and prevent diffusion out of the nucleus during digestion. Thus, following the 5 min digestion, the Incubation Buffer will contain very little pA-MNase digested chromatin. However, to be safe, the Incubation Buffer can be saved for potential future analysis. The addition of 200 μL STOP buffer will then chelate away remaining Ca++ and allow the digested chromatin fragments to freely diffuse out of the cells.
CRITICAL STEP: Heterologous spike-in DNA may be present in the STOP buffer to calibrate DNA amounts, for example to compare treatments or digestion time points. Alternatively, mapping to E. coli carry-over DNA from the pA-MNase and pA/G-MNase that gets fragmented during digestion suffices for calibration.
?. [Chromatin Digestion and Release Option 2: High Ca2+ / Low Salt (1 hour)]
Incubate 30 min @ 37 °C to release CUT&RUN fragments from the insoluble nuclear chromatin.
[Incubation]
[Incubation]
?. [Chromatin Digestion and Release Option 2: High Ca2+ / Low Salt (1 hour)]
Place on the magnet stand to clear. Cleanly transfer the supernatant containing digested chromatin to a fresh 1.5-mL microcentrifuge tube and proceed with Phenol Chloroform Extraction (Step 57).
[STOP w/ Chromatin]
NOTE: When performing CUT&RUN using untested antibodies, following removal of the STOP buffer w/ Chromatin it is a good idea to spin down and freeze the ConA bead-bound cells containing the insoluble nuclear chromatin that is left behind after MNase digestion. These cells may be useful for troubleshooting in cases where the user suspects chromatin solubility issues may be limiting yields. By preparing a sequencing library from the total DNA extracted from the "left-over" ConA bead-bound material  as described in Skene and Henikoff (eLife, 2017), and comparing the sequencing data, the user can then determine if the majority of the cleaved DNA was in fact being retained in the nuclear fraction.
?. [Chromatin Digestion and Release Option 1: Standard CUT&RUN (1.5 hours)]
Place on the magnet stand to clear. Cleanly transfer the supernatant containing digested chromatin to a fresh 1.5-mL microcentrifuge tube and proceed with Phenol Chloroform Extraction (Step 57).
[STOP w/ Chromatin]
NOTE: When performing CUT&RUN using untested antibodies, following removal of the STOP buffer w/ Chromatin it is a good idea to spin down and freeze the ConA bead-bound cells containing the insoluble nuclear chromatin that is left behind after MNase digestion. These cells may be useful for troubleshooting in cases where the user suspects chromatin solubility issues may be limiting yields. By preparing a sequencing library from the total DNA extracted from the "left-over" ConA bead-bound material  as described in Skene and Henikoff (eLife, 2017), and comparing the sequencing data, the user can then determine if the majority of the cleaved DNA was in fact being retained in the nuclear fraction.
?. [Chromatin Digestion and Release Option 3: Direct Ligation (1.5 hours)]
Remove liquid from the cap and side with a quick pulse on a micro-centrifuge. Place on the magnet stand to clear (~30 sec) and pull off all of the liquid.
NOTE: The CUT&RUN w/ Direct Ligation protocol is ideal for users who want to work quickly to prepare libraries, as it bypasses the phenol chloroform extraction and allows the user to potentially go from live cells to completed sequencing ready libraries in less than 12 hrs. This protocol is essentially a manual version of the AutoCUT&RUN protocol (https://www.protocols.io/view/autocut-run-genome-wide-profiling-of-chromatin-pro-ufeetje). Thus, samples processed manually using CUT&RUN w/ Direct Ligation are likley to be the most comparable to samples prepared using the automated format.
[Magnet stand]
?. [Chromatin Digestion and Release Option 3: Direct Ligation (1.5 hours)]
Place each tube at a low angle on the vortex mixer set to low (~1100 rpm) and add 24 μL of ice cold 1X pA-MNase Reaction Mix (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.
[1X pA-MNase Reaction Mix]
NOTE: Significantly reducing the volume during the MNase reaction and STOP allows the chromatin that is released into the supernatant to serve as input for End Repair and Adapter Ligation steps without requiring phenol chloroform extraction.
?. [Chromatin Digestion and Release Option 3: Direct Ligation (1.5 hours)]
Insert tubes into the 1.5 ml wells of a heater block sitting in wet ice to chill down to 0 °C. Incubate at 0 °C for the desired digestion time (default is 30 min).
[Incubation]
[Incubation]
CRITICAL STEP: MNase binds DNA but only cleaves when Ca++ is present, so that digestion is a zero-order reaction that seems to be less temperature-dependent than the subsequent diffusion of released pA-MNase-bound particles that can digest accessible regions of the genome. Cleavage and release of particles in most of the cell population can be obtained at 0 °C while minimizing background cleavages attributable to diffusion. We have found that digestion at ambient temperature or higher results in unacceptable background cleavage levels.
?. [Chromatin Digestion and Release Option 3: Direct Ligation (1.5 hours)]
Add 8 μL 4X STOP Buffer and mix by gentle vortexing. When there are multiple time points, remove 24 μL and add to a new tube containing 8 μL 4X STOP Buffer and mix by gentle vortexing.
[4X STOP Buffer]
CRITICAL STEP: Heterologous spike-in DNA may be present in the STOP buffer to calibrate DNA amounts, for example to compare treatments or digestion time points. Alternatively, mapping to E. coli carry-over DNA from the pA-MNase and pA/G-MNase that gets fragmented during digestion suffices for calibration.
?. [Chromatin Digestion and Release Option 3: Direct Ligation (1.5 hours)]
Incubate 30 min @ 37 °C to release CUT&RUN fragments from the insoluble nuclear chromatin.
[Incubation]
[Incubation]
?. [Chromatin Digestion and Release Option 3: Direct Ligation (1.5 hours)]
Place on the magnet stand to clear. Cleanly transfer 30 μL of the supernatant containing digested chromatin to a fresh 0.6 mL Low-Bind microcentrifuge tube, and immediately proceed with End Repair and Adapter Ligation (Step 70).
[STOP w/ Chromatin]
NOTE: When performing CUT&RUN using untested antibodies, following removal of the STOP buffer w/ Chromatin it is a good idea to spin down and freeze the ConA bead-bound cells containing the insoluble nuclear chromatin that is left behind after MNase digestion. These cells may be useful for troubleshooting in cases where the user suspects chromatin solubility issues may be limiting yields. By preparing a sequencing library from the total DNA extracted from the "left-over" ConA bead-bound material  as described in Skene and Henikoff (eLife, 2017), and comparing the sequencing data, the user can then determine if the majority of the cleaved DNA was in fact being retained in the nuclear fraction.
?. [PCR Enrichment of CUT&RUN Libraries (2-3 hours)]
Remove Ampure Bead Slurry from refrigerator, resuspend beads by vortexing and allow to equilibrate to room temperature before using.
NOTE: 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.
?. [PCR Enrichment of CUT&RUN Libraries (2-3 hours)]
Round 1: add 1.0 X the volume of Ampure Beads (e.g. to 85 µL sample add 85 µL Ampure Beads) and mix well by pipetting up and down 10 times. Incubate at room temperature for 5-10 min to allow beads to bind DNA.
[Ampure Beads]
[Incubation]
?. [PCR Enrichment of CUT&RUN Libraries (2-3 hours)]
Place on magnetic stand and allow to clear for 5 min. Without disturbing the beads remove liquid and discard.
[Magnet Stand]
[Liquid Waste]
?. [PCR Enrichment of CUT&RUN Libraries (2-3 hours)]
Without disturbing the beads add 200 µL 80% EtOH solution and incubate for 30 seconds.
[80% EtOH]
[Incubation]
?. [PCR Enrichment of CUT&RUN Libraries (2-3 hours)]
Leave the sample on the magnetic stand and remove and discard EtOH.
[EtOH Waste]
?. [PCR Enrichment of CUT&RUN Libraries (2-3 hours)]
Repeat steps 82-83 to wash the beads a second time with 80% EtOH. Then carefully remove residual EtOH, and air-dry beads for 5 min.
[Air-Dry]
?. [PCR Enrichment of CUT&RUN Libraries (2-3 hours)]
Remove tube from the magnet, add 50 µL 10 mM Tris-HCl pH 8, and pipette up and down 10X to resuspend beads. Incubate 5-10 min to allow DNA to elute off beads.
[10 mM Tris-HCl pH 8]
[Incubation]
?. [PCR Enrichment of CUT&RUN Libraries (2-3 hours)]
Prepare 25-50 mL of HXP Buffer (20% PEG 8000, 2.5 M NaCl), by mixing equal volumes of 40% PEG 8000 (wt/vl) and 5 M NaCl. HXP Buffer can be stored at 4 °C for up to two weeks.
NOTE: Once mixed with NaCl, PEG becomes light sensitive and slowly degrades over time.
?. [PCR Enrichment of CUT&RUN Libraries (2-3 hours)]
Round 2: To rebind the DNA to beads for a second clean up add 1.2 X the volume of HXP Buffer to DNA + beads (e.g. to 50 µL sample add 60 µL HXP Buffer) and mix well by pipetting up and down 10 times. Incubate at room temperature for 5-10 min to allow beads to bind DNA.
[HXP Buffer]
[Incubation]
?. [PCR Enrichment of CUT&RUN Libraries (2-3 hours)]
Place on magnetic stand and allow to clear for 5 min. Without disturbing the beads remove liquid and discard.
[Magnet Stand]
[Liquid Waste]
?. [PCR Enrichment of CUT&RUN Libraries (2-3 hours)]
Without disturbing the beads add 200 µL 80% EtOH solution and incubate for 30 seconds.
[80% EtOH]
[Incubation]
?. [PCR Enrichment of CUT&RUN Libraries (2-3 hours)]
Leave the sample on the magnetic stand and remove and discard EtOH.
[EtOH Waste]
?. [PCR Enrichment of CUT&RUN Libraries (2-3 hours)]
Repeat steps 89-90 to wash the beads a second time with 80% EtOH. Then carefully remove residual EtOH, and Air-Dry beads for 5 min.
[Air-Dry]
?. [PCR Enrichment of CUT&RUN Libraries (2-3 hours)]
Remove tube from the magnet, add 35 µL 10 mM Tris-HCl pH 8, and pipette up and down 10X to resuspend beads. Incubate 5-10 min to allow DNA to elute off beads.
[10 mM Tris-HCl pH 8]
[Incubation]
?. [PCR Enrichment of CUT&RUN Libraries (2-3 hours)]
Place on a magnetic stand and allow to clear for 3 min. Transfer the 30 µL of adapter ligated DNA to new 0.6-mL tube.
[Magnet Stand]
[Adapter ligated DNA]
?. [PCR Enrichment of CUT&RUN Libraries (2-3 hours)]
Prepare KAPA PCR Master Mix (~20 µL per sample):
?. [PCR Enrichment of CUT&RUN Libraries (2-3 hours)]
Add 20 µL KAPA PCR Master Mix to each sample and mix well by pipetting up and down 10 times.
[KAPA PCR Master Mix]
?. [PCR Enrichment of CUT&RUN Libraries (2-3 hours)]
Place in thermocycler and run the following program with heated lid:
CRITICAL STEP: To minimize the contribution of large DNA fragments, PCR cycles should be at least 12-14 cycles, preferably with a 10 s 60 °C combined annealing/extension step. Good results have been obtained with the Hyper-prep kit (KAPA Biosystems).
CRITICAL STEP: After PCR is complete, be extra careful to avoid contamination of any pre-PCR reagents with post-PCR products, which have P5 and P7 primer binding sites at either end and so even a minute amount of contamination in a solution or on a pipette tip can dominate the final library. Ideally, post-PCR manipulations should be done at a workbench that is never used for pre-PCR steps.
?. [PCR Enrichment of CUT&RUN Libraries (2-3 hours)]
Round 1: Add 1.1 X the volume of Ampure Beads (e.g. to 50 µL sample add 55 µL Ampure Beads) and mix well by pipetting up and down 10 times. Incubate at room temperature for 5-10 min to allow beads to bind DNA.
NOTE: To remove self-ligated adapter and left-over PCR reagents and prepare DNA libraries for sequencing, perform 2 more rounds of Ampure bead clean up.
[Ampure Beads]
[Incubation]
?. [PCR Enrichment of CUT&RUN Libraries (2-3 hours)]
Place on magnetic stand and allow to clear for 5 min. Without disturbing the beads remove liquid and discard.
[Magnet Stand]
[Liquid Waste]
?. [PCR Enrichment of CUT&RUN Libraries (2-3 hours)]
Without disturbing the beads add 200 µL 80% EtOH solution and incubate for 30 seconds.
[80% EtOH]
?. [PCR Enrichment of CUT&RUN Libraries (2-3 hours)]
Leave the sample on the magnetic stand and remove and discard EtOH.
[EtOH Waste]
?. [PCR Enrichment of CUT&RUN Libraries (2-3 hours)]
Repeat steps 99-100 to wash the beads a second time with 80% EtOH. Then carefully remove residual EtOH, and air-dry beads for 5 min.
[Air-Dry]
?. [PCR Enrichment of CUT&RUN Libraries (2-3 hours)]
Remove tube from the magnet, add 50 µL 10 mM Tris-HCl pH 8, and pipette up and down 10X to resuspend beads. Incubate 5-10 min to allow DNA to elute off beads.
[10 mM Tris-HCl pH 8]
[Incubation]
?. [PCR Enrichment of CUT&RUN Libraries (2-3 hours)]
Round 2: To rebind the DNA to beads for a second clean up add 1.2 X the volume of HXP Buffer to DNA + beads (e.g. to 50 µL sample add 60 µL HXP Buffer) and mix well by pipetting up and down 10 times. Incubate at room temperature for 5-10 min to allow beads to bind DNA.
[HXP Buffer]
[Incubation]
?. [PCR Enrichment of CUT&RUN Libraries (2-3 hours)]
Place on magnetic stand and allow to clear for 5 min. Without disturbing the beads remove liquid and discard.
[Magnet Stand]
[Liquid Waste]
?. [PCR Enrichment of CUT&RUN Libraries (2-3 hours)]
Without disturbing the beads add 200 µL 80% EtOH solution and incubate for 30 seconds.
[80% EtOH]
?. [PCR Enrichment of CUT&RUN Libraries (2-3 hours)]
Leave the sample on the magnetic stand and remove and discard EtOH.
[EtOH Waste]
?. [PCR Enrichment of CUT&RUN Libraries (2-3 hours)]
Repeat steps 105-106 to wash the beads a second time with 80% EtOH. Then carefully remove residual EtOH, and air-dry beads for 5 min.
[Air-Dry]
?. [PCR Enrichment of CUT&RUN Libraries (2-3 hours)]
Remove tube from the magnet, add 25-55 µL 10mM Tris-HCl pH 8, and pipette up and down 10X to resuspend beads. Incubate 5-10 min to allow DNA to elute off beads.
[10 mM Tris-HCl ph 8]
[Incubation]
?. [PCR Enrichment of CUT&RUN Libraries (2-3 hours)]
Place on a magnetic stand and allow to clear for 3 min. Transfer the 20-50uL of CUT&RUN DNA library to new 1.5 mL tube.
[Magnet Stand]
[CUT&RUN DNA library]
?. [Chromatin Digestion and Release Option 3: Direct Ligation (1.5 hours)]
Add 1 mL Dig-wash buffer, mix by inversion, or by gentle pipetting if clumps persist.
[Dig-wash buffer]
?. [Chromatin Digestion and Release Option 3: Direct Ligation (1.5 hours)]
Repeat Dig-wash steps 48-49.
[Repeat Dig-wash steps]
?. [Chromatin Digestion and Release Option 1: Standard CUT&RUN (1.5 hours)]
Remove liquid from the cap and side with a quick pulse on a micro-centrifuge. Place on the magnet stand to clear (~30 s) and pull off all of the liquid.
[Magnet stand]
?. [Chromatin Digestion and Release Option 3: Direct Ligation (1.5 hours)]
Remove liquid from the cap and side with a quick pulse on a micro-centrifuge. Place on the magnet stand to clear (~30 s) and pull off all of the liquid.
[Magnet stand]
?. [End Repair and Adapter Ligation (3 hours- overnight)]
Prepare 4X End Repair and A-tailing (ERA) buffer (10 µL/sample). Keep on Ice until use.
NOTE: Here we provide a version of the Henikoff lab in house library prep strategy for Illumina sequencing that uses TruSeq-Y Adapters with a free 3'T overhang. Alternatively, many users have also had success with the NEBNext Ultra I DNA Library Kit (E7645) following a protocl developed by Nan Liu in Stuart Orkin's lab (dx.doi.org/10.17504/protocols.io.wvgfe3w).
?. [End Repair and Adapter Ligation (3 hours- overnight)]
Add 10 µL 4X ERA buffer to each 30 µL sample and mix by pipetting up and down 5 times.
[4X ERA]
?. [End Repair and Adapter Ligation (3 hours- overnight)]
Place in a thermocycler that has been pre-cooled to 12 °C and run the following program with heated lid for temp >20 °C:
CRITICAL STEP: The 58 °C, 45 min dA-Tailing step is optimized to prevent short fragments (30-120bp) from denaturing, while still heat inactivating the exonuclease activity of the T4 DNA Polymerase.
?. [End Repair and Adapter Ligation (3 hours- overnight)]
Remove pre-annealed 0.15 µM TruSeq adapter from freezer and allow to thaw on ice.
?. [End Repair and Adapter Ligation (3 hours- overnight)]
Add 5 µL of 0.15 µM TruSeq adapter to each sample.
[Adapters]
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.
?. [End Repair and Adapter Ligation (3 hours- overnight)]
Prepare 2X Rapid Ligase solution (40 µL per sample). Keep on ice until use.
NOTE: Measure volumes carefully as the Enzymatics ligase buffer solution is very viscous. Prior to use pipette up and down 10X to ensure adequate mixing
?. [End Repair and Adapter Ligation (3 hours- overnight)]
Add 40 µL 2X Rapid Ligase solution to each sample and mix well by pipetting up and down 10 times.
[2X Rapid Ligase solution]
?. [End Repair and Adapter Ligation (3 hours- overnight)]
Place in a thermocycler that has been pre-cooled to 20 °C and incubate at 20 °C for 20-30 min. Following incubation proceed with Proteinase K digestion immediately.
?. [End Repair and Adapter Ligation (3 hours- overnight)]
Add 2 µL 10% SDS and 2 µL Proteinase K (20 mg/mL) to each sample. Mix well by vortexing and incubate at 37 °C for 1 hr-overnight.
[10% SDS]
[ProK (20mg/mL)]
[Incubation]
PAUSE POINT: The Proteinase K digest can proceed at 37 °C overnight. Also, if desired, the adapter ligated, de-proteinated DNA can then be frozen and stored at -20 °C for extended periods.
?. [Bind secondary antibody as required (15 min to 1.5 hours)]
Add secondary antibody to a final concentration of 1:100 or to the manufacturer’s recommended concentration for immunofluorescence.