TOP1-Cut&Tag_repeat3 (CRX2096228)
CUT&Tag-seq was performed on 9-day-old gTOP1a-4HA seedlings using the Hyperactive Universal CUT&Tag Assay Kit for Illumina Pro (Vazyme, TD904), with modifications based on a previously protocol63. Briefly, about 0.1 g of RTO tissue was firstly fixed in crosslinking buffer (1 percent formaldehyde, 0.4 M sucrose, 1 mM EDTA, 10 mM Tris-HCl pH 8.0, 1x protease inhibitor cocktail) for 10 min and stopped with 2 M glycine. Nuclei were isolated in the buffer (50 mM HEPES-KOH pH7.5, 150 mM NaCl, 1 mM EDTA, 1 percent Triton X-100, 0.1 percent sodium deoxycholate, 1 percent SDS) and then incubated with activated ConA beads, followed by overnight incubation with anti-HA antibody (Cell signaling Technology, C29F4) at 4 degrees Celsius in antibody buffer (20 mM HEPES pH 7.5, 0.5 mM spermidine, 150 mM NaCl, 2 mM EDTA, 1 mg/mL BSA, 1x protease inhibitor cocktail, 0.05 percent w/v digitonin). After secondary antibody binding, pA/G-Tn5 transposase (0.04 uM) was added for tagmentation at 37 degrees Celsius for 1 h in the buffer containing 20 mM HEPES pH 7.5, 0.3M NaCl, 0.5 mM spermidine, 10 mM MgCl2, 1x protease inhibitor cocktail and 0.05 percent w/v digitonin. DNA was extracted following the manufacturer's instructions. Sequencing libraries were prepared by adding adapters and amplifying the DNA, and then sequenced on the Illumina NovaSeq 6000 platform (GENOME, China).