Multiplexed DNA Affinity purification sequencing (multiDAP-seq) of flowering plants [Arabidopsis thaliana]

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.

本研究采用多重DNA亲和纯化测序（multiplexed DNA affinity purification sequencing, multiDAP-seq）技术，解析了10种开花植物基因组中360个转录因子（transcription factors, TFs）的全基因组结合图谱。我们将基因组DNA片段文库置于96孔板中，与体外表达的融合HaloTag（HaloTag）标签的转录因子进行结合实验，最终获得了近3000张全基因组结合图谱。该数据集可用于转录因子结合位点的识别与比较调控分析，不仅揭示了高度保守的转录因子-DNA相互作用，还发现了支撑植物物种分化的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从5种植物的多种组织中获取的单细胞核RNA测序（single nuclei RNA-seq）数据集进行整合，以探究转录因子在调控细胞类型特异性基因表达模式中的作用。相关的单细胞核RNA测序数据集已提交至生物项目编号（BioProject ID）PRJNA1262374。转录因子（TFs）经合成后被克隆至大肠杆菌（E. coli）质粒载体中，获得由T7聚合酶启动子（T7 polymerase promoter）驱动的带HaloTag标签的转录因子。使用PCR引物制备用于体外蛋白表达的线性表达模板。将各物种的基因组DNA剪切至约150 bp，通过连接单索引（仅含i5索引）的Illumina接头进行唯一条形码标记，经PCR扩增以去除天然修饰后进行混合。将每个混合文库均分至96个孔中，每个孔均包含结合于Halo磁珠的一种独特的体外表达转录因子。捕获得到的结合转录因子的DNA片段随后经PCR扩增，以添加孔特异性的i7 Illumina接头。每块板均设置阴性对照孔，使用以缓冲液替代转录因子线性蛋白表达模板制备的体外蛋白表达空白产物作为对照。测序完成后，通过联合i5与i7索引对文库进行解复用，最终为每个转录因子对应每个物种生成一个唯一的FASTQ文件。将这些序列比对至参考基因组，并以对应板的合并阴性对照背景作为对照进行峰识别，以确定每个基因组中的转录因子结合位点。