BET-seq: Binding energy topographies revealed by microfluidics and high-throughput sequencing.

Biophysical models of transcriptional regulation rely on energetic measurements of the binding affinities between transcription factors (TFs) and target DNA binding sites. Historically, assays capable of measuring TF-DNA binding affinities have been relatively low-throughput (measuring ~10 sequences in parallel) and have required significant specialized equipment, limiting their use to a handful of laboratories. Recently, we developed an experimental assay and analysis pipeline that allows measurement of binding energies between a single TF and up to 10 DNA species in a single experiment (Binding Energy Topography by sequencing, or BET-seq) (Le et al., 2018). BET-seq employs the Mechanically Induced Trapping of Molecular Interactions (MITOMI) platform to purify DNA bound to a TF at equilibrium followed by high coverage sequencing to reveal relative differences in binding energy for each sequence. While we have previously used BET-seq to refine the binding affinity landscapes surrounding high-affinity DNA consensus target sites, we anticipate this technique will be applied in future work toward measuring a wide variety of TF-DNA landscapes. Here, we provide detailed instructions and general considerations for DNA library design, performing BET-seq assays, and analyzing the resulting data.