Optimized DNA affinity purification sequencing determines relative binding affinity of transcription factors

DAP-seq is an in vitro method to analyze the relative binding affinity of transcription factors to DNA. It is a fast and scalable method and its application to plant transcription factors with a binary bound/not bound readout was first published in 2016 by O'Malley and colleagues. Since DAP-seq only requires the transcription factor protein and genomic DNA of a species, it can easily be applied to any species with DNA extraction protocols and available genome sequence resources. We present an optimized DNA Affinity Purification sequencing (DAP-seq) protocol for the relative quantification of protein-DNA interactions and a practical guide for data analysis. The desired transcription factor is expressed in vitro and fused to a tag, such as a HaloTag. Genomic DNA is fragmented and adapters are ligated, added to the purified TF::HaloTag protein, and unspecifically bound DNA is washed away. After the bound DNA is recovered, we add a quantification step which homogenizes library size and improves reproducibility. The expanded downstream bioinformatic analysis identifies transcription factor binding sites in the genome followed by analyses of replicate robustness by comparing three different peak height measures, control characteristics, and relative binding affinity.