Transcription initiates the formation of single-stranded DNA (ssDNA) regions within the genome, delineating transcription bubbles, a highly dynamic genomic process. Kethoxal-assisted single-stranded DNA sequencing (KAS-seq) utilizing N₃-kethoxal has emerged as a potent tool for mapping specific guanine positions in ssDNA on a genome-wide scale. However, the original KAS-seq method required the costly Accel-NGS Methyl-seq DNA library kit. This study introduces an optimized iteration of the KAS-seq technique, referred to as adapter-tagged KAS-seq (atKAS-seq), incorporating an adapter tagging strategy. This modification involves integrating sequencing adapters via complementary strand synthesis using random N9 tagging. Additionally, by harnessing the potential of ascorbic acid (ASC), recognized for inducing global epigenetic changes, we employed the atKAS-seq methodology to elucidate critical pathways influenced by short-term, high-dose ASC treatment. Our findings underscore that atKAS-seq enables rapid and precise analyses of transcription dynamics and enhancer activities concurrently. This method offers a streamlined, cost-efficient, and low-input approach, affirming its utility in probing intricate genomic regulatory mechanisms.

转录启动基因组内单链 DNA (ssDNA) 区域的形成，描绘转录气泡，这是一个高度动态的基因组过程。利用 N ₃ -kethoxal的酮醛辅助单链 DNA 测序 (KAS-seq)已成为在全基因组范围内绘制 ssDNA 中特定鸟嘌呤位置的有效工具。然而，最初的 KAS-seq 方法需要昂贵的 Accel-NGS Mmethyl-seq DNA 文库试剂盒。本研究介绍了 KAS-seq 技术的优化迭代，称为接头标记 KAS-seq (atKAS-seq)，结合了接头标记策略。这种修改涉及使用随机 N9 标签通过互补链合成来整合测序接头。此外，通过利用抗坏血酸 (ASC) 的潜力，我们采用 atKAS-seq 方法来阐明受短期、高剂量 ASC 治疗影响的关键途径。我们的研究结果强调，atKAS-seq 能够同时快速、精确地分析转录动态和增强子活性。该方法提供了一种精简、经济高效且低投入的方法，证实了其在探索复杂的基因组调控机制中的实用性。