Department of Chemistry, Department of Biochemistry and Molecular Biology, Institute for Biophysical Dynamics, The University of Chicago, Chicago, IL, USA.
Howard Hughes Medical Institute, The University of Chicago, Chicago, IL, USA.
Nat Protoc. 2022 Feb;17(2):402-420. doi: 10.1038/s41596-021-00647-6. Epub 2022 Jan 10.
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-kethoxal-assisted labeling. We developed N-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 N-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.
转录及其动态变化对于基因表达调控至关重要。然而，很少有方法可以用低输入材料和高时间分辨率直接读出转录活性。本方案描述了 KAS-seq，这是一种使用 N-酮醛辅助标记来捕获全基因组单链 DNA（ssDNA）谱的强大而敏感的方法。我们开发了 N-酮醛，这是酮醛的叠氮衍生物，在 37°C 下，它可以在 5-10 分钟内在活细胞中与 ssDNA 的脱氧鸟嘌呤碱基反应，从而可以捕获动态变化。标记 DNA 的后续生物素化通过无铜点击化学进行。总的来说，KAS-seq 程序包括 N-酮醛标记、DNA 分离、生物素化、片段化、亲和下拉、文库制备、测序和生物信息学分析。在文库构建之前的标记和富集可以在 3-4 小时内完成，适用于动物组织和低至 1000 个培养细胞。我们最近的研究表明，通过 KAS-seq 测量的 ssDNA 信号同时揭示了转录活跃的 RNA 聚合酶（Pol）II、转录增强子、RNA Pol I 和 Pol III 活性以及潜在的非规范 DNA 结构的动态变化，具有高分析灵敏度。除了实验方案外，我们还在这里介绍了 KAS-pipe，这是一种用于 KAS-seq 的用户友好的集成数据分析管道。
