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An Ultrahigh-throughput Microfluidic Platform for Single-cell Genome Sequencing
Published on: May 23, 2018
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BET-seq: Binding energy topographies revealed by microfluidics and high-throughput sequencing
Arjun K Aditham1, Tyler C Shimko2, Polly M Fordyce3
1Department of Bioengineering, Stanford University, Stanford, CA, United States; Stanford ChEM-H, Stanford University, Stanford, CA, United States.
Methods in Cell Biology
|November 27, 2018
Summary
We developed Binding Energy Topography by sequencing (BET-seq), a high-throughput method to measure transcription factor (TF)-DNA binding energies for millions of sequences. This technique significantly advances biophysical models of transcriptional regulation.
Area of Science:
- Molecular Biology
- Biophysics
- Genomics
Background:
- Biophysical models of transcriptional regulation require accurate measurements of transcription factor (TF)-DNA binding affinities.
- Traditional TF-DNA binding assays are low-throughput and require specialized equipment, limiting their widespread application.
Purpose of the Study:
- To provide detailed instructions and considerations for performing and analyzing Binding Energy Topography by sequencing (BET-seq) assays.
- To enable high-throughput measurement of TF-DNA binding energies for millions of DNA sequences in a single experiment.
Main Methods:
- Utilizes the Mechanically Induced Trapping of Molecular Interactions (MITOMI) platform to isolate TF-bound DNA at equilibrium.
- Employs high-coverage sequencing to quantify relative binding energies across a vast library of DNA sequences.
- Describes DNA library design, assay execution, and data analysis for BET-seq.
Main Results:
- BET-seq enables the measurement of binding energies for up to 10^6 DNA sequences per experiment, a significant increase in throughput.
- The method refines binding affinity landscapes around TF consensus sites.
- Provides a comprehensive guide for implementing and analyzing BET-seq data.
Conclusions:
- BET-seq is a powerful, high-throughput technique for characterizing TF-DNA binding landscapes.
- This method advances the study of transcriptional regulation by providing detailed energetic information.
- The detailed protocol facilitates broader adoption and application of BET-seq in research labs.
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