GSM9666514: Bulk CoCUT&Tag co-occupancy reads (R2) for H3K4me3 and H3K27me3 in K562 cells (A5).; 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 Bulk 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. Bulk CoCUT&Tag: Prior to starting the experiment, the anti-SunTag-Tn5 and anti-MoonTag-Tn5 were loaded with differentially barcoded MEDA&MEDB adapters, that are the same as the adapters used of sciCUT&Tag (Janssens et al. Nature Protocols 2024). These barcodes are essential to distinguish reads corresponding to protein 1 from reads targeting protein 2 and then co-occupancy reads. Briefly, the differentially barcoded MEDA&MEDB oligos are annealed to the universal reverse MED. They are then mixed a equimolar concentration and are suspended in anti-SunTag-Tn5 or anti-MoonTag-Tn5 solution, 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 and the loaded anti-SunTag-Tn5 and anti-MoonTag-Tn5 are storage at -20°C. Bangs ConA beads (Bangs Cat. No. BP531) were prepared for binding (5 μL per CoCUT&Tag sample) by transferring them to a 1.5 mL microfuge tube and adding 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 mL of binding buffer and then resuspended in 100 μL of binding buffer. Whole cells were thawed at room temperature and mixed. For each sample, 25K 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 containing Bangs ConA beads was added to the whole cell suspension in 3 increments of 33 μL, with mixing in between each addition. After 10 minutes of binding at room temperature, the sample was split into 500 uL low bind tubes, 1 tube for each sample, and the supernatant was removed and the suspension was brought up in 15 μL of Antibody Buffer (1 mL Wash Buffer, 4 μL 0.5M EDTA [2 mM]). 1.5 μL of appropriate mouse antibody and 1.5 μL of the appropriate rabbit antibody was added for overnight incubation at 4°C. Before starting the second day we prepared 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]). 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. In addition, we pre-blocked the SunTag and MoonTag scaffold reagents. For each sample, we mixed 20 μL of Wash Block Buffer with 2 μL of αRb-12X-SunTag-GFP and 2 μL of αMs-12X-MoonTag-GFP and incubated at room temperature for 30 minutes. The supernatant was removed from the cells, and 20 μL of the Block Tag mix was added to the cells for incubation at 4°C for 1 hour. Samples were washed twice in 100 μL of Wash Block Buffer and brought up in 15 μL of 300-Wash Block Buffer. 1.5 μL aSun-Tn5 loaded with barcoded MEDA&B were added for incubation at 4°C for 1 hour. Samples were washed twice in 100 μL of 300-Wash Block Buffer and brought up in 50 μL of Tagmentation buffer. The samples were placed in a thermocycler with a heated lid and tagmentation was performed at 37°C for 1 hour. Samples were washed twice in 100 μL of 300-Wash Block Buffer and brought up in 15 μL of 300-Wash Block Buffer. 1.5 μL aMoon-Tn5 loaded with barcoded MEDA&B were added for incubation at 4°C for 1 hour. Samples were washed twice in 100 μL of 300-Wash Block Buffer and brought up in 50 μL of Tagmentation buffer. The samples were placed in a thermocycler with a heated lid and tagmentation was performed at 37°C for 1 hour. 0.1% SDS Release Buffer was prepared (220 μL 10 mM TAPS, 25 μL 1% SDS, 5 μL Thermolabile Proteinase K). Samples were then washed once with 200 uL of 10 mM TAPS pH 8.5. This TAPS wash is to remove salt to prepare the sample for PCR, and so we made sure no excess liquid remained in the cap of the tube or on the walls. The supernatant was the removed and 5 μL of 0.1% SDS Release Buffer was added to each sample, ensuring that the beads are suspended in this small volume by flicking the tube or vortexing. The chromatin release step was performed by incubated at 1 hour for 37°C and 1 hour for 58°C in a thermocycler with a heated lid. Samples were quenched with an addition of 4 μL of 1.5% TritonX-10 solution. This was followed by addition of 24 µL of nanopore H20 to each samples, followed by 18 µL of PCR “Master Mix” (for each sample: 10 µL 5 X HiFi buffer, 5.5 µL ddH2O, 1.5 µL 10 mM dNTP, 1 µL KAPA HiFi Polymerase). Then add 2 µL of a barcoded Forward primer (10 µM) and 2 µL of a barcoded Reverse primer (10 µM) to each sample, ensuring that each sample has at least one distinctly barcoded primer. Mix by vortexing. PCR was conducted on a thermocycler with a heated lid using the profile: 58°C for 5 min (gap fill), 72°C for 5 min, 98°C for 45 seconds, followed by 14 cycles of (98°C for 15 seconds, 60°C for 10 seconds, 72°C for 1 seconds), ending with 72°C for 1 minute and hold at 12°C. Samples are then purified using a standard Ampure bead cleanup with a 1.3X size selection eluted in 30 μL Tris-HCl pH 8.0 and then run on a Tapestation and pooled at equimolar concentrations. The library pool was was then subjected to a 0.9x Ampure bead cleanup to remove the PCR product of “internal tagmentation” that results from one adapter tagmenting into another. Sequencing is performed on an Element Aviti or Illumina Platform using the appropriate custom primers.