Multiplexed DNA Affinity purification sequencing (multiDAP-seq) of flowering plants [Capsella rubella]

We applied multiplexed DNA affinity purification sequencing (multiDAP-seq) to profile the binding landscapes of 360 transcription factors (TFs) across ten flowering-plant genomes. Genomic DNA fragment libraries were assayed in 96-well plates against in vitro expressed HaloTag-fused TFs, generating nearly 3,000 genome-wide binding maps. These data provide a resource for identification of TF binding sites and comparative regulatory analyses, revealing deeply conserved TF-DNA interactions alongside lineage-specific rewiring events that underpin plant diversification. We also integreated these TF binding datasets with singe nuclei RNA-seq datasets produced from multiple tissues of five plant species to investigate the roles of TFs in driving cell type-specific gene expression patterns. The associated single nuclei RNA-seq datasets were submitted under BioProject ID PRJNA1262374. Transcription factors (TFs) were synthesized and cloned into an E. coli plasmid vector to generate Halo-tagged TFs driven by a T7 polymerase promoter. PCR primers were used to produce linear expression templates for in vitro protein production. Genomic DNA from each species was sheared to ~150 bp, uniquely barcoded by ligation of a single-indexed (i5 index only) Illumina adapter, PCR-amplified to erase native modifications, and pooled. Each pool was split into 96 wells, with each well containg a unique in vitro expressed transcription factor bound to magnetic Halo beads. Captured TF-bound DNA fragments were subsequently PCR amplified to add well-specific i7 Illumina adapters. Negative control wells were included on each plate using mock in vitro protein expression product, produced by using buffer in place of the linear TF protein expression templates. After sequencing, libraries were demultiplexed by the combined i5 and i7 indexed resulting in a unique fastq file per species species per TF. These were aligned to reference genomes and peaks were called against merged negative control backgrounds from the corresponding plate to identify TF binding sites in each genome.

我们运用多重DNA亲和纯化测序（multiplexed DNA affinity purification sequencing, multiDAP-seq）技术，对10个开花植物基因组内360个转录因子（transcription factors, TFs）的结合图谱进行了系统表征。我们针对体外表达的HaloTag融合转录因子，在96孔板中对基因组DNA片段文库开展亲和富集检测，最终获得近3000张全基因组结合图谱。这些数据可为转录因子结合位点的鉴定以及比较调控分析提供宝贵资源，同时揭示了支撑植物演化分化的深度保守TF-DNA相互作用与谱系特异性调控重连事件。
我们还将上述转录因子结合数据集与来自5个植物物种多种组织的单核RNA测序（single nuclei RNA-seq）数据集进行整合，以探究转录因子在驱动细胞类型特异性基因表达模式中发挥的功能。相关单核RNA测序数据集已提交至BioProject数据库，编号为PRJNA1262374。
研究人员将转录因子合成并克隆至大肠杆菌（E. coli）质粒载体中，构建了由T7聚合酶启动子（T7 polymerase promoter）驱动的HaloTag标记转录因子表达体系。通过PCR引物制备线性表达模板，用于体外蛋白合成。提取各物种的基因组DNA并剪切成约150 bp的片段，通过连接单索引（仅i5索引）Illumina适配器（Illumina adapter）进行唯一条码标记，经PCR扩增以去除天然修饰后混合文库。将每个混合文库均分至96孔板，每孔均加入结合于Halo磁珠的体外表达转录因子。随后对捕获到的结合有转录因子的DNA片段进行PCR扩增，以添加孔特异性的i7 Illumina适配器。每块板均设置阴性对照孔，采用模拟体外蛋白表达产物——即用缓冲液替代线性转录因子蛋白表达模板制备所得——作为对照。
测序完成后，通过联合i5与i7索引对文库进行解复用，最终为每个物种的每个转录因子生成唯一的fastq文件（fastq file）。将这些文件比对至参考基因组（reference genome），并以对应板的合并阴性对照背景为对照进行峰识别，以鉴定各基因组中的转录因子结合位点。