Kethoxal-assisted single-stranded DNA sequencing captures global transcription dynamics and enhancer activity in situ

Nat Methods. 2020 May;17(5):515-523. doi: 10.1038/s41592-020-0797-9. | PubMed

Tong Wu^1,2, Ruitu Lyu^1,2, Qiancheng You^1,2, Chuan He^3,4,5

1. Department of Chemistry, University of Chicago, Chicago, IL, USA.
2. Howard Hughes Medical Institute, University of Chicago, Chicago, IL, USA.
3. Department of Chemistry, University of Chicago, Chicago, IL, USA. chuanhe@uchicago.edu.
4. Howard Hughes Medical Institute, University of Chicago, Chicago, IL, USA. chuanhe@uchicago.edu.
5. Department of Biochemistry and Molecular Biology, Institute for Biophysical Dynamics, University of Chicago, Chicago, IL, USA. chuanhe@uchicago.edu.

Abstract

Transcription is a highly dynamic process that generates single-stranded DNA (ssDNA) in the genome as 'transcription bubbles'. Here we describe a kethoxal-assisted single-stranded DNA sequencing (KAS-seq) approach, based on the fast and specific reaction between N3-kethoxal and guanines in ssDNA. KAS-seq allows rapid (within 5 min), sensitive and genome-wide capture and mapping of ssDNA produced by transcriptionally active RNA polymerases or other processes in situ using as few as 1,000 cells. KAS-seq enables definition of a group of enhancers that are single-stranded and enrich unique sequence motifs. These enhancers are associated with specific transcription-factor binding and exhibit more enhancer-promoter interactions than typical enhancers do. Under conditions that inhibit protein condensation, KAS-seq uncovers a rapid release of RNA polymerase II (Pol II) from a group of promoters. KAS-seq thus facilitates fast and accurate analysis of transcription dynamics and enhancer activities simultaneously in both low-input and high-throughput manner.

Presented By Tong Wu