Marinov Georgi K, Greenleaf William J
Department of Genetics, School of Medicine, Stanford University, Stanford, CA, USA.
Department of Applied Physics, Stanford University, Stanford, CA, USA.
Bio Protoc. 2025 May 5;15(9):e5306. doi: 10.21769/BioProtoc.5306.
The KAS-ATAC assay provides a method to capture genomic DNA fragments that are simultaneously physically accessible and contain single-stranded DNA (ssDNA) bubbles. These are characteristic features of two of the key processes involved in regulating and expressing genes-on one hand, the activity of -regulatory elements (cREs), which are typically devoid of nucleosomes when active and occupied by transcription factors, and on the other, the association of RNA polymerases with DNA, which results in the presence of ssDNA structures. Here, we present a detailed protocol for carrying out KAS-ATAC as well as basic processing of KAS-ATAC datasets and discuss the key considerations for its successful application. Key features • Allows mapping of simultaneously accessible and ssDNA-containing DNA fragments. • Describes the execution of N3-kethoxal labeling and transposition of native chromatin. • Describes the pulldown of biotin-labeled DNA fragments and library generation. • Describes basic KAS-ATAC data processing steps.
KAS-ATAC分析提供了一种捕获基因组DNA片段的方法，这些片段同时在物理上是可及的并且包含单链DNA（ssDNA）气泡。这些是参与基因调控和表达的两个关键过程的特征——一方面是调控元件（cREs）的活性，其在活跃时通常不含核小体并被转录因子占据，另一方面是RNA聚合酶与DNA的结合，这会导致ssDNA结构的存在。在这里，我们展示了进行KAS-ATAC的详细方案以及KAS-ATAC数据集的基本处理，并讨论其成功应用的关键注意事项。关键特性 • 允许对同时可及且含ssDNA的DNA片段进行定位。 • 描述了N3-乙二醛标记和天然染色质转座的执行过程。 • 描述了生物素标记的DNA片段的下拉和文库生成。 • 描述了基本的KAS-ATAC数据处理步骤。
