TITLE: CUT&Tag@home
AUTHORS: Steven Henikoff, Derek Janssens, Hatice Kaya-Okur, Jorja Henikoff, Kami Ahmad

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">CUT&Tag@home uses a modification of Bench-top CUT&Tag which includes incubation in 0.1% SDS post-tagmentation for quantitative release of targeted fragments, followed directly by PCR with Triton-X100 to neutralize the SDS. This protocol is performed in single PCR tubes from nuclei to sequencing-ready libraries and should be suitable for high throughput.</div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/protocols-files/public/09f36cba505cc0a5e3c6dd075ecf5a93a45eb79426b7f42d8460df133f1e9904/b27titfe.png" /></div></div>

[STEPS]
?. [ REAGENT SETUP (for up to 16 samples)]
Binding buffer Mix 200 μL 1M HEPES-KOH pH 7.9, 100 μL 1M KCl, 10 μL 1M CaCl2 and 10 μL 1M MnCl2, and bring the final volume to 10 mL with dH2O. Store the buffer at 4 °C for up to several months.Wash buffer Mix 1 mL 1 M HEPES pH 7.5, 1.5 mL 5 M NaCl, 12.5 μL 2 M spermidine, bring the final volume to 50 mL with dH2O, and add 1 Roche Complete Protease Inhibitor EDTA-Free tablet. Store the buffer at 4 °C for up to several months.Antibody buffer Mix 4 μL 0.5 M EDTA and 10 µL 100X BSA with 1 mL Wash buffer and chill on ice.300-wash buffer Mix 1 mL 1 M HEPES pH 7.5, 3 mL 5 M NaCl and 12.5 μL 2 M spermidine, bring the final volume to 50 mL with dH2O and add 1 Roche Complete Protease Inhibitor EDTA-Free tablet. Store at 4 °C for up to several months.Tagmentation solution Mix 1 mL 300-wash buffer and 10 µL 1 M MgCl2 (to 10 mM).TAPS wash buffer Mix 1 mL dH2O, 10 µL 1 M TAPS pH 8.5, 0.4 µL 0.5 M EDTA (10 mM TAPS, 0.2 mM EDTA)0.1% SDS Release solution Mix 10 µL 10% SDS and 10 µL 1 M TAPS pH 8.5 in 1 ml dH2O0.67% Triton neutralization solution Mix 67 µL 10% Triton-X100 + 933 µL
?. [Prepare Concanavalin A-coated beads (15 min)]
Transfer 85 µL ConA bead slurry into 1 mL Binding buffer in a 1.5 mL tube and mix by pipetting. Place the tube on a magnet stand to clear (30 s to 2 min).
?. [Prepare Concanavalin A-coated beads (15 min)]
Resuspend and withdraw enough of the ConA bead slurry such that there will be 3-5 μL for each final sample of up to ~100,000 mammalian cells. The following is for 16 samples.
?. [Prepare Concanavalin A-coated beads (15 min)]
Withdraw the liquid completely, and remove from the magnet stand. Add 1 mL Binding buffer and mix by pipetting.
?. [Prepare Concanavalin A-coated beads (15 min)]
Place on magnet stand to clear, withdraw liquid, and resuspend in 85 µL Binding buffer (for 5 μL per sample).
?. [Bind nuclei to ConA bead (15 min)]
Thaw a frozen native or lightly cross-linked nuclei aliquot at room temperature, for example by placing in a 20 ml beaker of water.
We have observed reduced library yields with lightly cross-linked nuclei with some epitopes and antibodies.
?. [Bind nuclei to ConA bead (15 min)]
Mix 25-200 µL of nuclei suspension with 3-5 µL ConA beads in thin-wall 0.5 ml PCR tubes and let sit at room temperature for 10 min.
Using more than ~100,000 nuclei or >5 µL Con A beads per sample may inhibit the PCR.
?. [Bind nuclei to ConA bead (15 min)]
Place the tubes on a magnet stand to clear and withdraw the liquid.
In low-retention PCR tubes, surface tension will cause bead-bound cells to slide down to the bottom of the tube, so to avoid losses here and below, set the pipettor to 5 µL less than the liquid volume to be removed.
?. [Bind primary antibody (1 hr)]
Resuspend cells in 50 µL Antibody buffer then 0.5µL antibody (1:100) with gentle vortexing.
For bulk processing, resuspend in Antibody buffer containing antibody (1:100) with gentle vortexing.
We use 1:100 by default or the manufacturer’s recommended concentration for immunofluorescence.
?. [Bind primary antibody (1 hr)]
Place on a Rotator at room temperature and incubate 1-2 hr.
Volumes up to 50 µL will remain in the tube bottom by surface tension during rotation.
To evaluate success of the procedure without requiring library preparation, include in parallel a positive control antibody (e.g.α-H3K27me3), and an optional negative control by omitting the primary antibody.
?. [Bind secondary antibody (1 hr)]
Place tubes on the magnet stand to clear. Withdraw the liquid with the pipettor set to 5 µL less than the volume to be removed.
?. [Bind secondary antibody (1 hr)]
Mix the secondary antibody 1:100 in Wash buffer and squirt in 50 µL per sample while gently vortexing to allow the solution to dislodge the beads from the sides.
Although not needed for CUT&RUN, the secondary antibody step is required for CUT&Tag to increase the number of Protein A binding sites for each bound antibody. We have found that without the secondary antibody the efficiency is very low.
?. [Bind secondary antibody (1 hr)]
Place the tubes on a Rotator and rotate at room temperature for 30 min.
?. [Bind secondary antibody (1 hr)]
After a quick spin (
?. [Bind secondary antibody (1 hr)]
With the tubes still on the magnet stand, carefully add 500 µL Wash buffer. The surface tension will cause the beads to slide up along the side of the tube closest to the magnet.
?. [Bind secondary antibody (1 hr)]
Slowly withdraw the liquid with a 1 mL pipette tip without disturbing the beads.
To withdraw the liquid, set the pipettor to 600 µL, and keep the plunger depressed while lowering the tip to the bottom. The liquid level will rise to near the top completing the wash. Then ease off on the plunger until all the liquid is withdrawn, and remove the pipettor. This will leave behind at most a small drop of liquid.
?. [Bind pA-Tn5 adapter complex (1.5 hr)]
Mix pA-Tn5 adapter complex in 300-wash buffer to a final concentration of 1:200.
pA-Tn5 aliquots received from the CUT&RUN team are pre-loaded with adapters suitable for single- or dual-indexing on a paired-end Illumina flow-cell platform.
?. [Bind pA-Tn5 adapter complex (1.5 hr)]
Squirt in 50 µL per sample of the pA-Tn5 mix while vortexing and invert by rotation to allow the solution to dislodge most or all of the beads.
When using the recommended Macsimag magnet stand, dislodging the beads can be done by removing the plexiglass tube holder from the magnet, and with fingers on top to prevent the tubes from opening up or falling out, invert by rotating sharply a few times.
?. [Bind pA-Tn5 adapter complex (1.5 hr)]
After a quick spin (
?. [Bind pA-Tn5 adapter complex (1.5 hr)]
Place the tubes on a magnet stand to clear and pull off the liquid.
?. [Bind pA-Tn5 adapter complex (1.5 hr)]
With the tubes still on the magnet stand, carefully add 500 µL 300-wash buffer.
?. [Tagmentation and particle release (2.5 hr)]
Resuspend the bead/nuclei pellet in 50 µL tagmentation solution while vortexing or inverting by rotation to allow the solution to dislodge most or all of the beads as in Step 20.
?. [Tagmentation and particle release (2.5 hr)]
After a quick spin (
?. [Tagmentation and particle release (2.5 hr)]
Place tubes on a magnet stand, and withdraw the liquid, with the pipettor set to 5 µL less than the volume to be removed, followed by a quick spin.
?. [Tagmentation and particle release (2.5 hr)]
Place the tubes on a magnet stand and remove any remaining liquid using a 20 µL pipette tip, then resuspend the beads in 50 µL TAPS wash and invert by rotation to mix.
?. [Tagmentation and particle release (2.5 hr)]
Place the tubes on a magnet stand and remove any remaining liquid using a 20 µL pipette tip, and proceed immediately to the next step.
?. [Tagmentation and particle release (2.5 hr)]
Resuspend the beads in 5 µL 0.1% SDS Release solution using a fresh 20 µL pipette tip to dispense while wetting the sides of the tubes to recover the fraction of beads sticking to the sides.
Twirling the tube back and forth rapidly between thumb and finger will effectively wet the sides of the tube, followed by a quick spin to bring most of the beads to the bottom.
?. [Tagmentation and particle release (2.5 hr)]
Incubate at 58 ºC for 1 hr in a PCR cycler with heated lid to reverse the cross-links and release pA-Tn5 from the tagmented DNA.
?. [PCR (1 hr)]
To the PCR tube containing the bead slurry add 15 µL Triton neutralization solution + 2 µL of 10 µM Universal or barcoded i5 primer + 2 µL of 10 µM uniquely barcoded i7 primers, using a different barcode for each sample. Vortex on full and place tubes in metal tube holder on ice.
Indexed primers are described by Buenrostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523:486 (2015). Do not use Nextera or NEB primers.
?. [PCR (1 hr)]
Add 25 µL NEBnext (non-hot-start), vortex to mix, followed by a quick spin.
?. [PCR (1 hr)]
Mix, quick spin and place in Thermocycler and begin cycling program with heated lid:    Cycle 1: 58 °C for 5 min (gap filling)    Cycle 2: 72 °C for 5 min (gap filling)    Cycle 3: 98 °C for 30 sec    Cycle 4: 98 °C for 10 sec    Cycle 5: 60 °C for 10 sec    Repeat Cycles 4-5 11 times    72°C for 1 min and hold at 8 °C
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To minimize the contribution of large DNA fragments and excess primers, PCR should be performed for no more than 12 cycles, preferably with a 10 s 60-63 °C combined annealing/extension step.
The cycle times are based on using a conventional Peltier cycler (e.g., BioRad/MJ PTC200), in which the ramping times (3 °C/sec) are sufficient for annealing to occur as the sample cools from 98 °C to 60 °C. Therefore, the use of a rapid cycler with a higher ramping rate will require either reducing the ramping time or other adjustments to assure annealing.
Do not add extra PCR cycles to see a signal by capillary gel electrophoresis (e.g. Tapestation). If there is no nucleosomal ladder for the H3K27me3 positive control, you may assume that CUT&Tag failed, but observing no signal for a sparse chromatin protein such as a transcription factor is normal, and the barcoded sample can be concentrated for mixing with the pool of barcoded samples for sequencing. Extra PCR cycles reduce the complexity of the library and may result in an unacceptable level of PCR duplicates.
?. [Post-PCR Clean-up (30 min)]
After tubes have cooled, remove from the cycler and add 1.3 volume (65 µL) SPRI bead slurry, mixing by pipetting up and down.
?. [Post-PCR Clean-up (30 min)]
Quick spin and let sit at room temperature 5-10 min.
?. [Post-PCR Clean-up (30 min)]
Place on magnet 5 min to allow the beads to clear before withdrawing the liquid. While still on the magnet stand add 200 µL 80% ethanol.
?. [Post-PCR Clean-up (30 min)]
Withdraw the liquid with a pipette to the bottom of the tube, and add 200 µL 80% ethanol.
?. [Post-PCR Clean-up (30 min)]
Withdraw the liquid and after a quick spin, remove the remaining liquid with a 20 µL pipette. Do not air-dry the beads, but proceed immediately to the next step.
?. [Post-PCR Clean-up (30 min)]
Remove from the magnet stand, add 22 µL 10 mM Tris-HCl pH 8 and vortex on full. Let sit at least 5 min.
?. [Post-PCR Clean-up (30 min)]
Place on the magnet stand and allow to clear.
?. [Post-PCR Clean-up (30 min)]
Remove the liquid to a fresh 1.5 ml tube with a pipette.
?. [Tapestation analysis and DNA sequencing (outsource)]
Determine the size distribution and concentration of libraries by capillary electrophoresis using an Agilent 4200 TapeStation with D1000 reagents or equivalent.
?. [Tapestation analysis and DNA sequencing (outsource)]
Mix barcoded libraries to achieve equal representation as desired aiming for a final concentration as recommended by the manufacturer. After mixing, perform an SPRI bead cleanup if needed to remove any residual PCR primers.
?. [Tapestation analysis and DNA sequencing (outsource)]
Perform paired-end Illumina sequencing on the barcoded libraries following the manufacturer’s instructions. For maximum economy, paired-end PE25 is more than sufficient for mapping to large genomes.
Using paired-end 25x25 sequencing on a HiSeq 2-lane rapid run flow cell we obtain ~300 million total mapped reads, or ~3 million per sample when there are 96 samples mixed to obtain approximately equal molarity.
?. [Data processing and analysis]
We align paired-end reads to hg19 using Bowtie2 version 2.3.4.3 with options: --end-to-end --very-sensitive --no-unal --no-mixed --no-discordant --phred33 -I 10 -X 700. For mapping E. coli carry-over fragments, we also use the --no-overlap --no-dovetail options to avoid possible cross-mapping of the experimental genome to that of the carry-over E. coli DNA that is used for calibration. Tracks are made as bedgraph files of normalized counts, which are the fraction of total counts at each basepair scaled by the size of the hg19 genome.To calibrate samples in a series for samples done in parallel using the same antibody we use counts of E. coli fragments carried over with the pA-Tn5 the same as one would for an ordinary spike-in. Our sample script in Github can be used to calibrate based on either a spike-in or E. coli carry-over DNA.
?. [Data processing and analysis]
Most data analysis tools used for ChIP-seq data, such as bedtools, Picard and deepTools, can be used on CUT&Tag data. Analysis tools designed specifically for CUT&RUN/Tag data include the SEACR peak caller also available as a public web server, CUT&RUNTools and henipipe.
See https://www.biorxiv.org/content/10.1101/2020.04.15.043083v1.full.pdf for examples of CUT &Tag@home applications
?. [Bind secondary antibody (1 hr)]
After a quick spin, place the tubes on a magnet stand to remove the last drop with a 20 µL pipette tip and proceed immediately to the next step.
?. [Bind pA-Tn5 adapter complex (1.5 hr)]
Slowly withdraw the liquid with a 1 mL pipette tip as in Step 17.
?. [Bind pA-Tn5 adapter complex (1.5 hr)]
Squirt in 50 µL per sample of 300-wash buffer while vortexing and invert by rotation to allow the solution to dislodge most or all of the beads.
?. [Bind pA-Tn5 adapter complex (1.5 hr)]
After a quick spin, place the tubes on a magnet stand to clear and withdraw the liquid, with the pipettor set to 5 µL less than the volume to be removed.
?. [Bind pA-Tn5 adapter complex (1.5 hr)]
After a second quick spin, place the tubes on a magnet stand to remove the last drop with a 20 μL pipette tip and proceed immediately to the next step.
?. [Tagmentation and particle release (2.5 hr)]
Place tubes on a magnet stand, and withdraw the liquid with the pipettor set to 5 µL less than the volume to be removed, followed by a quick spin.
?. [Bind secondary antibody (1 hr)]
After a quick spin, replace on the magnet stand and withdraw the last drop with a 20 µL pipette tip.