GSM9666730: Single-cell CoCUT&Tag co-occupancy reads (R1) for H3K4me2 and H3K27me3 (S48) in bone marrow mononuclear cells; Homo sapiens; OTHER
Cells were bound to ConA beads followed by antibody binding and binding of antigen-peptide repeat nanobody fusions, then binding of barcoded anti-SunTag-Tn5 and tagmentation in 300 mM Wash buffer, and binding of barcoded anti-MoonTag-Tn5 and tagmentation in 300 mM Wash buffer, this was followed by the direct to PCR method Single-cell CoCUT&Tag Protocol: Prepare 50 g/L Casein Block Buffer, pH 7.0: Add 5.99 g NaOH to 400 mL of deionized water. Slowly add 25 grams of Casein while stirring to avoid clumping. Incubate overnight at 37°C while shaking at 250 RPM to ensure that the protein fully dissolves. Adjust pH to 7.0 by adding 12 N HCl dropwise with a pH meter, with heavy stirring to avoid precipitation. Adjust to 500 mL. Flash freeze in liquid nitrogen and store at -80°C. Single-cell CoCUT&Tag: Prior to starting the experiment, 96-well plates containing differentially barcoded anti-SunTag-Tn5 complexes and 96-well plates containing differentially barcoded anti-MoonTag-Tn5 complexes were prepared using a similar protocol to the one described previously for sciCUT&Tag and sciCUT&Tag2in1 (Janssens et al. Nature Protocols 2024; Janssens et al. Cell Reports 2026). The only difference is that for single-cell CoCUT&Tag, after distributing the differentially barcoded annealed MEDA&MEDB oligos across the 96 well plate, they are suspended in anti-SunTag-Tn5 or anti-MoonTag-Tn5 solution, and the plate is sealed, vortexed to mix, and loading is allowed to proceed for 1.5 hrs at room temperature. An equal volume of 90% glycerol is then added to each well with a multichannel pipette and the solution is split between multiple 96 well plates for storage at -20°C. We typically start with 1-2 million cells, but the protocol can be run with fewer starting cells the risk being the nanowell chip may need to be loaded with fewer than 10 cells per well. Regardless of the starting cell number, Magnefy ConA beads (Bangs Cat. No. MFY531) were prepared for binding by mixing the solution and adding 200 µL to 500 µL of Binding Buffer (400 μL of 1 M HEPES-KOH pH 7.9 [20 mM], 200 μL of 1 M KCl [10 mM] 20 μL of 1 M CaCl2 [1 mM], and 20 μL of 1 M MnCl2 [1 mM], adjusted to 20 mL nanopure H2O). The mixture was added to a magnet stand and the supernatant removed. The beads were washed with another 500 µL of binding buffer and then resuspended in 100 μL of binding buffer. Whole cells were thawed at room temperature and mixed to a final count of 1-2 million. Cells were added to a 1.5 mL tube and centrifuged at 600 x g for 3 minutes.  The supernatant was removed and the pellet was resuspended in 900 mL of Wash Buffer (48 mL ddH2O, 1 mL 1M HEPES pH 7.5[20 mM], 1.5 mL 5M NaCl [150 mM], 12.5 µL 2M Spermidine [0.5 mM], 250 μL of 10% Triton X-100 [0.05%],1 Roche Complete EDTA-free protease tablet). The binding buffer was added to the whole cell suspension in 3 increments of 33 μL additions, with mixing in between each addition. After 10 minutes of binding at room temperature, the supernatant was removed and the suspension was brought up in 500 μL of Wash buffer. The cells were lightly crosslinked by adding 3.2 μL of 16% Formaldehyde (Thermo Scientific Cat no. 28906) to a final concentration of 0.1%. After 2 minutes, the reaction was quenched by an addition of 15 μL of 2.5 M glycine. The samples were washed in 200 uL of Wash buffer and brought up in 100 μL of Antibody Buffer (1 mL Wash Buffer, 4 μL 0.5M EDTA [2 mM]). At this point the sample could be split for multiple conditions, but here 10 μL of the appropriate mouse antibody and 10 μL of the appropriate rabbit antibody was added for overnight incubation at 4°C. Wash Block Buffer (48 mL ddH2O, 1 mL 1M HEPES pH 7.5[20 mM], 1.5 mL 5M NaCl [150 mM], 12.5 µL 2M Spermidine [0.5 mM], 250 μL of 10% Triton X-100 [0.05%],0.25 g BSA (Sigma-Aldrich Cat no. A9647), 5 mL 50 g/L mL Casein Block Buffer pH 7.0, 1 Roche Complete EDTA-free protease tablet), 300-Wash Block Buffer (48.5 mL Wash Block Buffer, 1.5 mL 5M NaCl [300 mM NaCl]) and TAPS buffer (500 μL 1M TAPS pH 8.5 [10 mM] in 49.5 mL ddH2O), and Tagmentation Buffer (9.9 mL 300-Wash Block Buffer, 100 μL 1 M MgCl2 [10 mM]) were prepared. Samples were washed twice in 100 μL of Wash buffer and suspended in 50 μL of Wash Block Buffer for 30 minutes at room temperature. 120 μL of Wash Block Buffer was mixed with 12 μL of αRb-12X-SunTag-GFP and 12 μL of αMs-12X-MoonTag-GFP and incubated at room temperature for 30 minutes. The supernatant was removed from the cells, and the Block SunTag/MoonTag mix was added to the cells for incubation at 4°C for 1 hour. If multiple antibody combinations are to be run in parallel, split the 120 μL of Block SunTag/MoonTag mix between these samples. Samples were washed twice in 100 μL of Wash Block Buffer and resuspended in 1500 μL of 300-Wash Block Buffer. Here, if there are multiple antibody combinations, bring each sample up in a volume of 300-Wash Block Buffer proportional to the fraction of that plate dedicated to that sample. Next, 15 μL of sample was added to each well of a 96-well plate (Eppendorf, cat. no. 0030129504) with frequent mixing in between additions to prevent bead bound cells from settling to the bottom of the tube. 1.5 μL from a differentially barcoded αSun-Tn5 MEDA&B PAGE plate were added to each well with a multi-channel pipette, and the plate was sealed and incubated at 4°C for 1 hour. The plate of cells was spun down for one second at 1,200 RPM and placed on a 96-ring low elution magnet (ALPAQU cat. no. A000350). The wells were washed two times with 100 μL of 300-Wash Block Buffer and brought up in 50 μL of Tagmentation buffer. The plate was sealed and placed on a thermocycler, and tagmentation was performed at 37°C for 1 hour. The plate was then placed back on the magnet stand, washed twice with 100 μL of 300-Wash Block Buffer and brought up in 15 μL of wash block buffer. 1.5 μL of differentially barcoded αMoon-Tn5 MEDA&B PAGE plate was added to each well with a multi-channel pipette and the plate was sealed and incubated at 4°C for 1 hour. The plate of cells was spun down for one second at 1,200 RPM and placed back on the 96-well magnet stand. The wells were washed two times with 100 μL of 300-Wash Block Buffer and brought up in 50 μL of Tagmentation buffer. The plate was sealed and placed on a thermocycler, and tagmentation was performed at 37°C for 1 hour. Tagmentation Buffer was removed from each well on the magnet stand, the plate was transferred to a non-magnetic rack, and all wells were washed and combined using 8 aliquots of 100 μL of Wash Buffer via multichannel pipetting each row together. Another round of 8 x100 μL was used to clean up the rows and the total 1.6 mL was combined and placed in a tube on a magnet stand. The pool was washed once with 500 μL of Wash Buffer and resuspended in 500 μL of Wash Buffer. For size filtering, the 500 μL of Wash Buffer was passed through a 20μm filter (pluriSelect Cat no. 43-10020-40) at 100xg for 3 minutes. DAPI staining was performed by adding 10 μL of 10,000× DAPI diluted 2:198 in TAPS Buffer. Nuclei were counted on a Countess slide. Based on the cell concentration, cells were diluted to 10 cells per 35 nL in 10 mM TAPS, and 2:198 DAPI was added to a final concentration of 1x. Once in TAPS buffer cells will start to lyse and clump, and so it is important to start the dispense on the ICELL8 quickly. 80 μL were added into wells A1-D2 on the 384 source plate and 35 nL were dispensed into each well of a ICELL8 350v chip. The chip was then dried with a paper wipe, sealed, spun for 1 minute at 1,200 x g and imaged. 0.19% SDS Release Buffer was prepared (616 μL 10 mM TAPS, 152 μL 1% SDS, 32 μL Thermolabile Proteinase K). 35 nL of 0.19% SDS + thermolabile proteinase K in TAPS was dispensed on the chip, which was dried, sealed, and spun in the same manner. The chip was then incubated at at 37°C for 1 hour and then 58°C for 1 hour. The chip was spun again, and 35 nL of 2.5% Triton X-100 was dispensed, and the chip was dried, sealed, and spun in the same manner. 35 nL i5 primer was dispensed, and the chip was dried, sealed, and spun in the same manner. 35 nL i7 primer was dispensed, and the chip was dried, sealed, and spun in the same manner. KAPA PCR Mix was prepared with 450 μL 5 X HiFi buffer, 247.5 μL ddH2O, 67.5 μL 10 mM dNTP, 45 μL KAPA HiFi polymerase. 100 nL of KAPA PCR Mix was dispensed on the chip, and the chip was dried, sealed, and spun in the same manner. The chip was then placed into the custom thermocycler, and PCR was performed using the following thermal profile: 58°C for 5 min (gap fill), 72°C for 10 min, 98°C for 45 seconds, followed by 13 cycles of (98°C for 15 seconds, 60°C for 10 seconds, 72°C for 5 seconds), ending with 72°C for 1 minute and hold at 12°C. After amplification the chip was spun in a collection kit for 2 minutes at 1,200 RPM. The DNA pool was then put through a 1.3x Ampure bead cleanup and eluted in 100 μL Tris-HCl pH 8.0. This was followed by a 0.9x Ampure bead cleanup to remove the PCR product of “internal tagmentation” that results from one adapter tagmenting into another. DNA was eluted in 25 μL Tris-HCl pH 8. Final libraries were analyzed on a TapeStation. Sequencing is performed on an Element Avidi or Illumina Platform using the appropriate custom primers.