KAS-seq: genome-wide sequencing of single-stranded DNA by N3-kethoxal-assisted labeling

Transcription and its dynamics are crucial for gene expression regulation. However, very few methods can directly read out transcriptional activity with low-input material and high temporal resolution. This protocol describes KAS-seq, a robust and sensitive approach for capturing genome-wide single-stranded DNA (ssDNA) profiles using N-3-kethoxal-assisted labeling. We developed N-3-kethoxal, an azido derivative of kethoxal that reacts with deoxyguanosine bases of ssDNA in live cells within 5-10 min at 37 degrees C, allowing the capture of dynamic changes. Downstream biotinylation of labeled DNA occurs via copper-free click chemistry. Altogether, the KAS-seq procedure involves N-3-kethoxal labeling, DNA isolation, biotinylation, fragmentation, affinity pull-down, library preparation, sequencing and bioinformatics analysis. The pre-library construction labeling and enrichment can be completed in as little as 3-4 h and is applicable to both animal tissue and as few as 1,000 cultured cells. Our recent study shows that ssDNA signals measured by KAS-seq simultaneously reveal the dynamics of transcriptionally engaged RNA polymerase (Pol) II, transcribing enhancers, RNA Pol I and Pol III activities and potentially non-canonical DNA structures with high analytical sensitivity. In addition to the experimental protocol, we also introduce here KAS-pipe, a user-friendly integrative data analysis pipeline for KAS-seq. KAS-seq is an N-3-kethoxal-assisted single-stranded DNA sequencing approach that allows rapid, sensitive and genome-wide mapping of single-stranded DNA produced in situ using as few as 1,000 cells or animal tissues.

Automated summary

This article introduces a method called KAS-seq, which captures genome-wide single-stranded DNA profiles using N-3-kethoxal-assisted labeling. This method allows for rapid, sensitive and genome-wide mapping of single-stranded DNA produced in situ, and can simultaneously detect multiple transcriptional activities as well as non-canonical DNA structures.