Multiplexed DNA Affinity purification sequencing (multiDAP-seq) of flowering plants [Sorghum bicolor]

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.