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

Area of Science:

• Molecular Biology
• Genomics
• Epigenetics

Background:

• Transcription dynamics are vital for gene expression regulation.
• Existing methods lack the sensitivity and temporal resolution for low-input material.
• Directly measuring transcriptional activity remains a challenge.

Purpose of the Study:

• To introduce KAS-seq, a novel method for genome-wide ssDNA profiling.
• To enable high-resolution capture of dynamic transcriptional events.
• To provide a user-friendly analysis pipeline (KAS-pipe) for KAS-seq data.

Main Methods:

• KAS-seq utilizes N3-kethoxal for rapid ssDNA labeling in live cells.
• Copper-free click chemistry facilitates downstream biotinylation.
• The protocol includes DNA isolation, fragmentation, affinity pull-down, and sequencing.

Main Results:

• KAS-seq captures genome-wide ssDNA profiles with high sensitivity and temporal resolution.
• The method accurately reflects transcriptionally engaged RNA polymerase II activity.
• KAS-seq reveals dynamics of enhancers, RNA Pol I, Pol III, and potential non-canonical DNA structures.

Conclusions:

• KAS-seq offers a robust and sensitive approach for studying transcription dynamics.
• The protocol is efficient, requiring only 3-4 hours for labeling and enrichment.
• KAS-seq is applicable to various sample types, including low cell numbers and animal tissues.