Screen of ZaNAC25 targets by DNA-affinity purification sequencing (DAP-Seq)
DAP-seq analysis were conducted in Bluescape Scientific CO.,LTD (China).Firstly, ZaNAC25 was inserted into the HaloTag expression vector .The fusion protein was expressed in the TNT SP6 Coupled Wheat Germ Extract System and purified using Magne Halo Tag Beads. Then, the mixed tissues including leaf, stem, flower and fruit were collected for DNA library preparations. Finally, the DNA libraries were constructed using the NEXTFLEX? Rapid XP DNA-Seq Kit. The protein-bound beads were incubated with 50 ng of adapter-ligated gDNA fragments, then the bound DNA fragments were released into solution. After PCR product purification and selection, the DNA fragments were sequenced on an Illumina NavoSeq. Libraries obtained from beads without ZaNAC25 protein addition were set as negative control.