TITLE: AutoCUT&Tag: streamlined genome-wide profiling of chromatin proteins on a liquid handling robot
AUTHORS: Derek Janssens, Steven Henikoff

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>The CUT&Tag method is based on antibody tethering of the Tn5 transposase to profile the genome-wide occupancy of DNA-binding proteins, histone modifications and chromatin modifying proteins </span><span style = "font-style:italic;">in situ</span><span> starting from relatively low cell numbers (1-100K cells). Activation of tethered transposase integrates library adapters at the sites of bound chromatin factors. By taking advantage of protocol modifications that enable Tn5 release and PCR in the same sample reaction chamber, we adapted CUT&Tag to an automated 96 well format. We provide programs and equipment specifications for performing AutoCUT&Tag on a Beckman Coulter Biomek FX liquid handling robot equipped for magnetic separation and temperature control, but in principle the method could be easily translated to other liquid handling units. Using this automated protocol a single operator is able to generate up to 96 CUT&Tag libraries in a single day that are ready to be pooled and sequenced.</span></div></div>

[STEPS]
?. [Prepare Nuclei]
Obtain cells of interest and centrifuge at 600XG for 3 min in a 1.5 mL microfuge tube (if prepping nuclei from ≤ 1 million cells) or in a 15 mL conical tube if preparing  cells in bulk.
?. [Prepare Nuclei]
Carefully remove the liquid and resuspend cells in 1 mL cold NE 1 Buffer per 1 million cells by gentle pipetting or flicking the tube.
?. [Prepare Nuclei]
Incubate on ice for 10 min to lyse the cell membrane and release the nuclei.
?. [Prepare Nuclei]
Centrifuge at 600XG for 3 min and carefully remove the liquid.
?. [Prepare Nuclei]
Resuspend nuclei in 1 mL Wash Buffer per 1 million cells.
?. [Prepare Nuclei]
Optional: To lightly cross-link the nuclei add 16% formaldehyde to 0.1% (e.g. 6.25 µL 16% formaldehyde to 1 mL of Wash Buffer). Stop cross-linking after 2 min by addition of 2.5 M Glycine to a final concentration of 75 mM (e.g. 30 µL of 2.5 M Glycine to 1 mL of Wash Buffer).
?. [Prepare Nuclei]
Centrifuge at 1200XG for 3 min and carefully remove the liquid.
?. [Prepare Nuclei]
Resuspend lightly cross-linked nuclei in 1 mL Wash Buffer per 1 million cells.
?. [Bind nuclei to beads and bind primary antibody]
While gently vortexing the nuclei (~1100 rpm), add 5-10 µL of the Concanavalin A coated-bead suspension per sample in a dropwise manner. Note: Using more than ~100,000 nuclei or >10 µL Con A beads per sample may inhibit the PCR.
?. [Bind nuclei to beads and bind primary antibody]
Place on tube nutator at room temperature for 5-10 min.
?. [Bind nuclei to beads and bind primary antibody]
Mix well by vigorous inversion to ensure the bead-bound nuclei are in a homogenous suspension and divide into aliquots in 0.6-mL low-bind tubes, one for each antibody to be used.
?. [Bind nuclei to beads and bind primary antibody]
Place on the magnet stand to clear and pull off and discard the liquid.
?. [Bind nuclei to beads and bind primary antibody]
Resuspend ConA bead bound nuclei in 100 µL of the Antibody Buffer per sample. Tap to dislodge the beads that are stuck to the side.
?. [Bind nuclei to beads and bind primary antibody]
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).
?. [Bind nuclei to beads and bind primary antibody]
Place on the tube nutator at room temperature for 1-2 hr.
?. [AutoCUT&Tag Reaction]
If Wash Buffer was prepared more than 24 hrs prior, add fresh Roche Complete Protease Inhibitor tablets (1/50 mL) and allow to dissolve (~5 min). Then prepare the following solutions: CRITICAL STEP: If a primary antibody was used from any species other than Rabbit, an additional Secondary Antibody Solution will need to be prepared to include 125 µL per sample (1:100 dilution of secondary antibody). Make sure the secondary antibody that is used is compatible with Protein-A binding (e.g. Rabbit anti-Mouse IgG works well because Protein A has a high affinity for Rabbit IgG, however Goat anti-Mouse IgG is problematic because Protein A has little to no affinity for Goat IgG).         Note: Store Wash Buffer, Secondary Antibody Solution, 300 Wash Buffer, pA-Tn5 Solution, and Tagmentation Buffer on ice. Keep 0.1 % SDS Release Buffer, and 10 mM TAPS Buffer pH 8.5 at Room Temp.
?. [AutoCUT&Tag Reaction]
Remove the seal and lid and place a fresh box of AP96 200 µL Tips on the Tip Loading ALP at position TL1 on the Biomek Deck. Place the ALPAQUA Magnet Plate at position P3 and the ALPAQUA LE Magnet Plate at position P6 on the Biomek Deck. Place an empty labeled Deep Well Plate at position P9 on the Biomek Deck for collecting liquid waste. Set the Heating/Cooling Unit to 37oC.
?. [AutoCUT&Tag Reaction]
Select the Pre-Programmed AutoCUT&Tag Project on the Biomek and open the CUT&Tag Full Reaction Method file (described in the BEFORE STARTING and Guidelines sections).
?. [AutoCUT&Tag Reaction]
Transfer Wash Buffer into a reservoir and dispense 1 mL into each of the active wells of a labeled Deep Well Plate using a Multi-Channel Pipette and place at position P8 on the Biomek deck. Transfer 300 Wash Buffer into a reservoir and dispense 1 mL into each of the active wells of a labeled Deep Well Plate using a Multi-Channel Pipette and place at position P7 on the Biomek deck.
?. [AutoCUT&Tag Reaction]
Transfer Tagmentation Buffer into a reservoir and dispense 125 µL into each of the active wells of a labeled V-Bottom Plate using a Multi-Channel Pipette and place at position P5 on the Biomek deck (Bottom). Transfer pA-Tn5 Solution into a reservoir and dispense 125 µL into each of the active wells of a labeled V-Bottom Plate using a Multi-Channel Pipette and stack on top of the Tagmentation Buffer V-Bottom Plate at position P5 on the Biomek deck (Middle). Transfer Secondary Antibody Solution into a reservoir and dispense 125 µL into each of the active wells of a labeled V-Bottom Plate using a Multi-Channel Pipette and stack on top of the pA-Tn5 Solution V-Bottom Plate and Tagmentation Buffer V-Bottom Plate at position P5 on the Biomek deck (Top).
?. [AutoCUT&Tag Reaction]
Transfer 0.1% SDS Release Buffer into a reservoir and dispense 125 µL into each of the active wells of a labeled V-Bottom Plate using a Multi-Channel Pipette and place at position P4 on the Biomek deck (Bottom). Transfer 10 mM TAPS Buffer pH 8.5 into a reservoir and dispense 125 µL into each of the active wells of a labeled V-Bottom Plate using a Multi-Channel Pipette and stack on top of the 0.1 % SDS Release Buffer V-Bottom Plate at position P4 on the Biomek deck (Top).
?. [AutoCUT&Tag Reaction]
Using wide bore 200 µL tips, resuspend Concanavalin A bead-bound nuclei + 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 P11 on the Biomek deck.
?. [AutoCUT&Tag Reaction]
Start the CUT&Tag Full Reaction Method on the Biomek.
?. [Chromatin Release and PCR Amplification]
Upon completion of the CUT&Tag Full Reaction program, immediately remove the PCR 96 Well Plate containing the ConA bead bound tagmented nuclei in 10 µL 0.1% SDS Release Buffer. Seal with an AlumaSeal 96 cover, and place in a ThermoCycler set to 58oC with heated lid for 1 hr.
?. [Chromatin Release and PCR Amplification]
During the 1 hr 58oC incubation thaw out the 10 µM i5/i7 PCR Primer Plate and PCR Reagents and prepare the following solutions:Note: To allow 3' extension of the tagmented DNA at the start of the PCR, avoid using HOT START polymerase.
?. [Chromatin Release and PCR Amplification]
Upon completion of the 1 hr 58oC incubation, remove the PCR 96 Well Plate containing the samples and allow to cool to room temp (~ 5 min). Then remove and discared the AlumaSeal 96 Cover. Transfer 0.15 % Triton X-100 Solution into a reservoir and using a Multi-Channel Pipette dispense and mix 54 µL into each sample in the PCR 96 Well Plate (pipette up and down 5-10 times).
?. [Chromatin Release and PCR Amplification]
Uncover the 10 µM i5/i7 PCR Primer Plate while being careful to avoid cross-contamination of barcoded adapters between wells. Using a Multi-Channel Pipette dispense and mix 4 µL 10 µM i5/i7 PCR Primer solution into each sample in the PCR 96 Well Plate (pipette up and down 5-10 times). Then reseal and freeze the 10 µM i5/i7 PCR Primer Plate.
?. [Chromatin Release and PCR Amplification]
Transfer KAPA PCR Master Mix into a reservoir and using a Multi-Channel Pipette dispense and mix 36 µL into each sample in the PCR 96 Well Plate (pipette up and down 5-10 times).
?. [Chromatin Release and PCR Amplification]
Seal the PCR 96 Well Plate plate using an AlumaSeal 96 Cover, place in thermocycler and run the following program with heated lid:Note: 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. 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 and extension to occur. Therefore, the use of a rapid cycler with a higher ramping rate will require either reducing the ramping time or other adjustments to ensure annealing and extension.PAUSE POINT: PCR amplified Libraries can be held at 8oC overnight or stored at 4oC indefinitely.
?. [Post-PCR DNA Cleanup]
Select the Pre-Programmed AutoCUT&Tag Project on the Biomek and open the CUT&Tag Post-PCR Cleanup Method file (described in the BEFORE STARTING and Guidelines sections).
?. [Post-PCR DNA Cleanup]
Remove Ampure Bead Slurry from refrigerator, resuspend beads by vortexing and allow to equilibrate to room temperature. Using a Reservoir and Multi-Channel Pipette distribute 130 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]
Upon completion of the PCR Amplification Reaction, remove the PCR 96 Well Plate containing the samples and allow to cool to room temp (~ 5 min). Then remove and discard the AlumaSeal 96 Cover and stack on a PCR Plate Rack positioned at P5 on the Biomek deck.
?. [Post-PCR DNA Cleanup]
Using a Reservoir and Multi-Channel Pipette 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. 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]
Remove the seal and Lid and place a fresh box of 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&Tag DNA Libraries at position P8 on the Biomek deck.
?. [Post-PCR DNA Cleanup]
Start the CUT&Tag Post-PCR Cleanup Method.
?. [Sequencing]
Upon completion of the CUT&Tag Post-PCR Cleanup program, remove the PCR 96 Well Plate containing clean CUT&Tag Libraries (position P8) from the Biomek deck and seal with an AlumaSeal 96 cover for subsequent analysis.Note: The excess liquid should be emptied from V-Bottom and Deep Well Plates. This labware can then be thoroughly washed with DI water (fill and rinse 5-10 times), allowed to air dry overnight, and then stored for reuse in subsequent experiments. PAUSE POINT: Libraries can be stored at 4oC indefinitely.
?. [Sequencing]
Determine the size distribution and concentration of libraries by Agilent 4200 TapeStation analysis.? TROUBLESHOOTING
?. [Sequencing]
Pool indexed CUT&Tag libraries and perform paired-end Illumina sequencing following the manufacturer’s instructions.
?. [Data Processing and Analysis]
We align paired-end reads using Bowtie2 version 2.4.1 with options: --end-to-end --very-sensitive --no-mixed --no-discordant -q --phred33 -I 10 -X 700. For mapping E.Coli 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.? TROUBLESHOOTING