GSM9666477: Standard CUT&Tag for H3K4me2 (Sigma Aldrich 07-030) in RS411 cells; Homo sapiens; OTHER
Cells were bound to ConA beads followed by antibody and secondary binding, followed by pA-Tn5 binding and tagmentation in 300 mM Wash buffer, this was followed by the direct to PCR method Standard CUT&Tag Protocol: CUT&Tag was performed using the direct to PCR protocol as previously described (PMID: 32913232). Briefly, whole cells were spun down at 600 X G and brought up in 500 µL Wash Buffer containing Triton-X 100 for permeabilization (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 tablet). ConA magnetic beads (Bangs, cat. no. BP531) were prepared by washing 5 µL per sample, 2 times with 500 µL 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], bring up to 20 mL with nanopure water).  ConA Beads were resuspended in 100 µL of Binding Buffer and were added to the whole cell suspensions in 3-33 µL increments, the sample was mixed by inversion after each addition. Bead binding was allowed to proceed for 10 min at room temperature, and the supernatant was removed on a magnet stand. Then cells were washed once with 500 µL of Wash Buffer and brought up in 15 µL of Antibody Buffer per CUT&Tag reaction/replicate. 1.5 µL of the rabbit or mouse primary antibody (e.g, rabbit-anti H3K27me3; Cell Signaling Technologies Cat No. 9733S). Antibody binding was allowed to proceed overnight at 4°C. The next day, cells were washed twice with 200 µL of Wash Buffer and brought up in 25 µL of Wash Buffer and 1.5 µL of secondary antibody (either guinea pig anti-rabbit IgG antibody; Antibodies Online Cat No. ABIN101961 or rabbit ant-mouse IgG antibody; Abcam Cat No. Ab46540) is added to each tube and incubation is allowed to proceed at 4°C for one hour. Cells were then washed twice with 200 µL of Wash Buffer and brought up in 15 µL of 300-Wash Buffer (48 mL ddH₂O, 1 mL 1M HEPES pH 7.5 [20 mM], 3 mL 5M NaCl [300 mM], 12.5 µL 2M Spermidine [0.5 mM], 250 µL of 10% Triton-X-100 [0.05%], 1 Roche Complete EDTA-free tablet) and 1.5 µL of pA-Tn5 purified and loaded according to standard protocols (dx.doi.org/10.17504/protocols.io.8yrhxv6) was added and incubation is allowed to proceed at 4°C for one hour. Cells were then washed twice with 200 µL of Wash Buffer and brought up in 50 µL of tagmentation buffer (1 mL 300-Wash Buffer + 10 µL 1M MgCl₂ [10 mM]), and tagmentation was carried out at 37 °C for 1 hr on a thermocycler with a heated lid. Cells were then washed one time on the magnet stand with 200 µL Wash Buffer and one time with TAPS Buffer (500 µL 1M TAPS pH 8.5 [10 mM] in 49.5 mL ddH₂O), and then brought up in 5 µL SDS Release Buffer (220 µL 10 mM TAPS, 25 µL 1% SDS [0.1% final], 5 µL Thermolabile Proteinase K). Samples are incubated on the thermocycler with a heated lid at 37°C for 1 hr and 58 degrees for one hour. The SDS is then quenched by addition of 4 uL of 1.5% Triton-X-100 (75 µL of 10% TritonX-100 solution in 425 µL of nano pure H20). Each sample is then diluted by addition of 24 µL of nanopore H20, 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 distinct barcode. Mix by vortexing. Then place samples in Thermocylcer and run the following protocol with a heated lid at 105°C: 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 and then run on a Tapestation and pooled at equimolar concentrations for sequencing on the Element Avidi Platform.