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

Nat Protoc. 2022 Feb;17(2):402-420. doi: 10.1038/s41596-021-00647-6. Epub 2022 Jan 10.

Abstract

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 N3-kethoxal-assisted labeling. We developed N3-kethoxal, an azido derivative of kethoxal that reacts with deoxyguanosine bases of ssDNA in live cells within 5-10 min at 37 °C, allowing the capture of dynamic changes. Downstream biotinylation of labeled DNA occurs via copper-free click chemistry. Altogether, the KAS-seq procedure involves N3-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.

Publication types

  • Review

MeSH terms

  • Animals
  • Biotinylation
  • Click Chemistry / methods
  • DNA, Single-Stranded* / chemistry
  • DNA, Single-Stranded* / genetics
  • DNA, Single-Stranded* / metabolism
  • Humans
  • Mice
  • Sequence Analysis, DNA / methods

Substances

  • DNA, Single-Stranded