GSM8328202: Dome H3K4me1 Triptolide rep 2; Danio rerio; OTHER
The CUT&RUN procedure was adapted from Hainer et al (Hainer and Fazzio, 2019), which incorporates optimizations of the method of Skene and Henikoff (Skene and Henikoff, 2017). Embryos were subjected to either mechanical yolk separation or chemical yolk lysis. For chemical yolk lysis, embryos were transferred to 1.5 mL Eppendorf tubes removing excess liquid with a P200 pipettor, then yolk lysis buffer added (55 mM NaCl,1.8 mM KCl, 1.25 mM NaHCO3). Tubes were shaken at 1100 RPM for 5' at room temperature (Eppendorf ThermoMixer C), centrifuged at 300xg for 30s to pellet, yolk lysis buffer drawn off, and 1 mL Yolk Lysis Wash Buffer was added (110 mM NaCl, 3.5 mM KCl, 2.7 mM CaCl2, 10 mM Tris pH 8.5). Tubes were shaken at 1100g for 2 minutes at room temperature, centrifuged at 300g to pellet, and supernatant was again removed and replaced with Nuclear Extraction Buffer (20 mM HEPES-KOH, pH 7.9, 10 mM KCl, 500 µM spermidine, 0.1% Triton X-100, 20% glycerol). For manually de-yolking, yolks were pinched off using Dumont #5 forceps in Ca-free Ringer's Solution (116 mM NaCl, 2.9 mM KCl, 5 mM HEPES, ph 7.2). Deyolked embryonic cells were transferred via Pasteur pipette into Nuclear Extraction Buffer taking care to transfer as little liquid as possible, mixed with gentle pipetting with a P200 pipettor, and then centrifuged at 600xg at 4ºC for 3 min. The free nuclei were then bound to 150 µL of activated concanavalin A beads (Polysciences #86057) at RT for 10mins. Nuclei were blocked for 5 min (5 mL Wash Buffer with 2 mM EDTA) at RT then incubated in 1:100 dilution of primary antibody for 2 hr at 4ºC, washed with Wash Buffer, incubated in a 1:200 dilution of pAG-MNase (gift from Sarah Hainer) for 1 hr at 4ºC, and washed again with Wash Buffer. The bound pAG-MNase was activated with 2 mM CaCl2 and allowed to digest for 30 mins at 0ºC, then stopped using 2x STOP buffer (200 mM NaCl, 20 mM EDTA, 4 mM EGTA, 50 µg/mL RNase A, 40 µg/mL glycogen). Yeast mononucleosomes were added to 2x STOP buffer at 10 pg/mL (untreated samples) or 20 pg/mL (triptolide/dmso samples) as a spike-in. Nuclei were incubated at 37ºC for 20 min followed by centrifuging for 5 min at 16,000xg, drawing off the DNA fragments with the supernatant. The extracted fragments were treated with SDS and proteinase K at 70ºC for 10 min followed by phenol chloroform extraction. Purified DNA was resuspended in 50 µL of water and verified by Qubit dsDNA high sensitivity and Fragment Analyzer. CUT&RUN libraries were constructed using the NEB Ultra II DNA library prep kit (NEB #E7645) according to manufacturer's protocol. DNA was end repaired and then ligated to sequencing adaptors diluted 1:100. Ligated DNA was purified with 0.9x AmpureXP beads and PCR amplified for 15 cycles, then purified again with 0.9x AmpureXP beads. Libraries were run on a 1.5% TBE agarose gel, and a band corresponding to 175 - 650 bp was cut out and gel purified using the NEB Monarch DNA gel extraction kit (#T1020). Concentration was verified by Qubit dsDNA high sensitivity and Fragment Analyzer.