Department of Chemistry, University of Chicago, Chicago, IL, USA.
Howard Hughes Medical Institute, University of Chicago, Chicago, IL, USA.
Nat Methods. 2020 May;17(5):515-523. doi: 10.1038/s41592-020-0797-9. Epub 2020 Apr 6.
Transcription is a highly dynamic process that generates single-stranded DNA (ssDNA) in the genome as 'transcription bubbles'. Here we describe a kethoxal-assisted single-stranded DNA sequencing (KAS-seq) approach, based on the fast and specific reaction between N-kethoxal and guanines in ssDNA. KAS-seq allows rapid (within 5 min), sensitive and genome-wide capture and mapping of ssDNA produced by transcriptionally active RNA polymerases or other processes in situ using as few as 1,000 cells. KAS-seq enables definition of a group of enhancers that are single-stranded and enrich unique sequence motifs. These enhancers are associated with specific transcription-factor binding and exhibit more enhancer-promoter interactions than typical enhancers do. Under conditions that inhibit protein condensation, KAS-seq uncovers a rapid release of RNA polymerase II (Pol II) from a group of promoters. KAS-seq thus facilitates fast and accurate analysis of transcription dynamics and enhancer activities simultaneously in both low-input and high-throughput manner.
转录是一个高度动态的过程，它在基因组中产生单链 DNA（ssDNA）作为“转录泡”。在这里，我们描述了一种基于 N-酮醛与 ssDNA 中鸟嘌呤之间快速而特异反应的酮醛辅助单链 DNA 测序（KAS-seq）方法。KAS-seq 允许使用少至 1000 个细胞，快速（5 分钟内）、灵敏且全基因组捕获和定位转录活性 RNA 聚合酶或其他过程原位产生的 ssDNA。KAS-seq 能够定义一组单链的增强子，这些增强子富含独特的序列基序。这些增强子与特定的转录因子结合，并表现出比典型增强子更多的增强子-启动子相互作用。在抑制蛋白质凝聚的条件下，KAS-seq 揭示了 RNA 聚合酶 II（Pol II）从一组启动子中快速释放。因此，KAS-seq 能够以高通量和低输入的方式同时快速、准确地分析转录动力学和增强子活性。
