GSM5569852: CUT&RUN_H3K4me3millipore_293T; Homo sapiens; OTHER
The ProteinA-MNase (pA-MNase) was purified in E. coli cells as described previously using a protocol adapted from Schmidt et al, 2014. Parental and UBR7KO 293T cells or Drosophila Kc167 spike-in cells were harvested at RT and resuspended in Wash Buffer (20 mM HEPES-NaOH pH 7.5, 150 mM NaCl, 0.5 mM spermidine, 1x Roche protease inhibitor cocktail) and counted using a Vi-Cell Blu cell counter. For each antibody, 4e5 293T cells were mixed with 1e5 Kc167 cells, then incubated for 7m with 10uL of Concanavalin A coated magnetic beads (BioMag) in Binding Buffer (20 mM HEPES-KOH pH 7.9, 10 mM KCl, 1 mM CaCl2, 1 mM MnCl2). The bead-cell slurry was permeabilized in Wash Buffer supplemented with 0.005% digitonin (“Digitonin Buffer”) as determined by optimization, then incubated with anti-H3K4me3 antibody, or IgG as a control, (1:50) in Antibody Buffer (20 mM HEPES-NaOH pH 7.5, 150 mM NaCl, 0.5 mM spermidine, 0.005% digitonin, 2 mM EDTA, 0.1% BSA, 100 nM TSA, 0.1 unit/mL citrate synthase, 1 mM Oxaloacetic acid, 1x Roche protease inhibitor cocktail) for 2 h at RT with nutation. The beads were washed twice in Digitonin Buffer, then incucbated in 100uL Digintonin buffer with 700 ng/mL pA-MNase for 1 h at 4°C with nutation. The beads were washed twice in Digitonin Buffer, then resuspended in a low-salt Wash Buffer (20 mM HEPES-NaOH pH 7.5, 0.5 mM spermidine, 0.005% digitonin, 1x Roche protease inhibitor cocktail) and chilled to 0°C. The beads were then incubated with ice-cold Calcium Incubation Buffer (3.5 mM HEPES-NaOH pH 7.5, 10 mM CaCl2, 0.005% digitonin) for 30 m. The beads were washed with EGTA-Stop Buffer (170 mM NaCl, 20 mM EGTA, 0.005% digitonin, 50 µg/mL RNase A) for 30m at 37°C to release the chromatin fragments. To extract the DNA, 200ul of Oligo Binding Buffer (Zymo) was added to each sample and the total volume was then added to a Zymo-Spin DCC-5 column and centrifuged. The column was washed twice with DNA Wash buffer from the DNA Clean & Concentrator-5 Kit (Zymo). The DNA was eluted from the column in 15ul of DNA Elution Buffer and sample concentration was measured using a Qubit 3.0 fluorometer and Qubit dsDNA HS Assay Kit. Illumina libraries were prepared using a NEBNext Ultra II Library Prep Kit for Illumina and NEBNext® Multiplex Oligos for Illumina® (Dual Index Primers Set 1) using a protocol adapted from the manufacturer. Modifications to the protocol are as follows: The End Prep heat inactivation step was conducted at 50 °C for 60 minutes. The NEBNext Adaptor was diluted 1:25 in 10 mM Tris-HCl, pH 8.0 with 10 mM NaCl. After Adaptor Ligation, a DNA cleanup using 1.75x volumes of Sera-Mag Select beads was conducted. The PCR cycle conditions were programmed as following: 1 cycle: 98° C for 30 seconds, 12 cycles: 98° C for 10 seconds, 65° C for 30 seconds, 1 cycle: 65° C for 5 minutes, Hold: 4° C. After PCR amplification, DNA cleanup was conducted twice with 1.2x volumes of Sera-Mag Select (Cytiva) beads. Quality control was conducted on the resulting 15ul elution using Agilent 4200 TapeStation D1000 ScreenTape to determine sample concentration and sample quality.