greenCUT&RUN of GFP before treatment with 1mM SA
Six leaves from two plants were collected before and after treatment with 1 mM SA for 4 h and stored at -80 C. Frozen samples were ground to a fine powder and dissolved in 15 mL of the lysis buffer (20 mM Tris-HCl pH 7.5, 20% glycerol, 20 mM KCl, 2 mM EDTA, 2.5 mM MgCl2, 8.56% sucrose, and the protease inhibitor cocktail). Samples were filtered sequentially through a 70-m filter and a 40-m filter before centrifuged at 1,500 x g at 4 C for 10 min. The pellet was resuspended in the nuclei isolation buffer (20 mM Tris-HCl pH 7.5, 20% glycerol, 2.5 mM MgCl2, 0.2% Triton X-100, and the protease inhibitor cocktail) and centrifuged at 1,500 x g at 4 C for 10 min. The above resuspension and centrifugation steps were repeated 4x, until the pellet was free of any green color. The pellet was resuspended in 1 mL of the greenCUT&RUN wash buffer (20 mM HEPES-KOH pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, and the protease inhibitor cocktail). Isolated nuclei were then bound to 40 L of Concanavalin A beads resuspended in 10 L of binding buffer (20 mM HEPES-KOH pH 7.5, 10mM KCl, 1 mM CaCl2, 1 mM MnCl2, and the protease inhibitor cocktail) and rotated for 10 min at room temperature. The beads were collected using a magnetic rack, the supernatant was then removed, the bound nuclei were then resuspended in 1 mL of the EDTA buffer (20 mM HEPES-KOH pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, 2 mM EDTA, and the protease inhibitor cocktail), and rotated at room temperature for 10 min. The beads were collected again and resuspended in 100 L of the greenCUT&RUN wash buffer containing 10 g/mL of nanobody-MNase and rotated at 4 C for 30 min. After rotation, beads were collected and washed twice in the greenCUT&RUN wash buffer. Beads were then put on ice, resuspended in 150 L of the calcium buffer (20 mM HEPES-KOH pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, 3 mM CaCl2, and the protease inhibitor cocktail) and incubated on ice for 30 min. After incubation, 100 L of the 2X stop buffer (340 mM NaCl, 20 mM EDTA, 10 mM EGTA, 100 g/mL RNase A, and 50 g/mL Glycogen) was added to the beads and incubated at 37 C for 30 min. After incubation, beads were removed, and the supernatant was collected for DNA isolation. 2 L of 10% SDS and 20 g of Proteinase K were added to the collected supernatant and incubated at 50 C for 1 h. Equal volume of Phenol:Chloroform:Isoamyl Alcohol (25:24:1, v/v) was added to the samples followed by vortexing. The solution was transferred to a phase lock tube and centrifuged for 5 min at 16,000 x g at room temperature. After centrifugation, equal volume of chloroform was added, samples were inverted 10x, and centrifuged for 5 min at 16,000 x g at room temperature. The top aqueous layer was then taken and moved into new tubes containing 3 L of 2 mg/mL of glycogen. 2x volumes of 100% ethanol was added to each sample to facilitated DNA precipitation overnight at -20 C. After DNA precipitation, samples were centrifuged for 10 min at 16,000 x g at 4 C. The supernatant was removed, the pellet was washed in 1 mL of 100% ethanol, and centrifuged for 5 min at 16,000 x g at 4 C. The supernatant was removed, and the pellet was air dried for 5 to 10 min. The pellet was resuspended in 50 L of H2O and used for library preparation. greenCUT&RUN libraries were constructed using the KAPA HyperPrep Kit (Roche Holding AG), with minor modifications. Briefly, end repair and A-tailing were performed at 20 C for 30 min followed by deactivation of the A-tailing enzyme at 58 C for 1 h. 1/100 diluted Illumina TruSeq DNA UD Indexes were ligated on to A-tailed DNA at 20 C for 30 min. Post-ligation cleanup was performed twice, first using 1x library volume of AMPure Beads, next with 1.2x library volume of AMPure Beads, followed by a double-sided size selection to remove larger DNA fragments and smaller adapter dimers, respectively, using 0.7X-1.2X library volume of AMPure Beads following the manufactures protocol (Roche Holding AG). Ligated libraries were then amplified using PCR and cleaned up twice with 1.2x library volume of AMPure Beads to generate final purified libraries. Library size and concentration were determined using Agilent 2100 Bioanalyzer and Qubit (Thermo Fisher Scientific), respectively.