DNA Affinity Purification Sequencing (DAP-Seq)

The genomic DNA (gDNA) of M5-90 was extracted using a Genomic DNA Extraction Kit (Zoobio, Nanjing, China). Purified gDNA was fragmented to 100–500 bp via sonication for 17 min at 20% amplitude and keeping it for 30 s on and 30 s off on ice. The size of the fragmented gDNA was checked using 1.5% agarose gel. DNA end-repair and dA-tailing were performed on 1 μg of fragmented gDNA via the NEXTflex Rapid DNA-Seq Kit (Zoobio, Nanjing, China). Ligation of end-repaired and adenylated DNA to DAP-adaptor (Mich Scientific, Beijing, China) was performed using the NEXTflex TM Enzyme Mix (Zoobio, Nanjing, China) according to the manufacturer´s instructions.

Dap-seq experiments were performed in duplicate, and beads in negative control group were incubated without protein. Ten microliters of Halo-tag magnetic beads (Yeasen, Shanghai, China) were washed three times in a 1.5 mL tube with 600 μL of sterilized PBS containing 0.01% Tween 20 (binding buffer). Twenty-five microliters of wheat cell-free expressed protein was added to 25 μL of binding buffer (containing washed beads) and incubated for 1 h on a rotating wheel at 25°C. Next, 50 μL of binding buffer was added and used to wash the bead-protein complexes five times. The supernatant was discarded, 25 μL of binding buffer (containing 100 μM MnCl₂) and an equal volume of the gDNA library were added, and then the mixture was placed on a rotating wheel for further incubation at 25°C for 1 h. A volume of 50 μL of binding buffer was added and used to wash the formed bead-protein-DNA complexes five times to remove unbound DNA. Finally, 30 μL of 50 mM Tris-HCl, with pH 8.5 was added to suspend the complexes, and incubated for 10 min at 98°C. After incubation, the samples were cooled at 4°C for 5 min, removing the beads, and the released DNA was collected and stored at -20°C for PCR amplification as described previously [40]. The sample was sequenced using a different indexed pair of primers.