Screen of LmMYB111 targets by DNA-affinity purification sequencing (DAP-Seq)

DAP-seq analysis were conducted in Bluescape Scientific CO.,LTD (China).Firstly, LmMYB111 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 flowers at different developmental stages 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 LmMYB111 protein addition were set as negative control.

DNA亲和纯化测序（DAP-seq）分析由中国蓝景科学有限公司（Bluescape Scientific CO.,LTD）完成。首先，将LmMYB111插入至HaloTag表达载体（HaloTag）中。所得融合蛋白在TNT SP6偶联麦胚提取物系统（TNT SP6 Coupled Wheat Germ Extract System）中表达，并通过Magne HaloTag磁珠（Magne Halo Tag Beads）进行纯化。随后，收集不同发育阶段的花组织以用于DNA文库制备。最后，使用NEXTFLEX® Rapid XP DNA测序试剂盒（NEXTFLEX? Rapid XP DNA-Seq Kit）构建DNA文库。将结合有LmMYB111蛋白的磁珠与50 ng的接头连接基因组DNA片段进行孵育，随后将结合的DNA片段洗脱至溶液中。经PCR产物纯化与片段筛选后，将所得DNA片段在Illumina NavoSeq测序平台上进行测序。将未添加LmMYB111蛋白的磁珠所制备的文库设为阴性对照。