Kethoxal-assisted single-stranded DNA sequencing (KAS-seq) for capturing transcription dynamics and enhancer activity

Abstract We describe a rapid and sensitive labeling of single-stranded DNA for sequencing (KAS-seq), based on a kethoxal-guanine reaction. KAS-seq procedure involves N 3 -kethoxal labeling, DNA isolation, biotinylation, fragmentation and enrichment, library preparation and sequencing. The whole process can be finished in a day. KAS-seq enables rapid measurement of the dynamics of transcriptionally-engaged Pol II, transcribing enhancers, Pol I and Pol III activities, and non-canonical DNA structures involving ssDNA simultaneously in situ , by using as few as 1,000 cells or mice tissues. The robust and tissue-friendly nature coupled with low input material requirement make KAS-seq a method that can be broadly applied. This step-by-step protocol is related to the publication “Kethoxal-assisted single-stranded DNA sequencing captures global transcription dynamics and enhancer activity in situ ” in Nature Methods .