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

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 N(3)-kethoxal and guanines in ssDNA in live cells and mouse tissues. KAS-seq enables rapid (within 5 min), sensitive, and genome-wide capture and mapping of ssDNA produced by transcriptionally active RNA polymerases or other processes in situ by using as few as 1,000 cells. KAS-seq defines a group of enhancers that are single-stranded, which enrich unique sequence motifs and are associated with specific transcription factor binding and more enhancer-promotor interactions. Under protein condensation inhibition conditions, KAS-seq uncovers a rapid release of RNA polymerase II (Pol II) from a group of promotors. KAS-seq thus facilitates fast, comprehensive, and accurate analysis of transcription dynamics and enhancer activities simultaneously in a low input and high-throughput manner.