DNA
生物素化
计算生物学
生物
DNA测序
增强子
抄写(语言学)
分子生物学
化学
遗传学
基因
基因表达
语言学
哲学
作者
Ruitu Lyu,Tong Wu,Allen Zhu,Diana West-Szymanski,Xiaocheng Weng,Mengjie Chen,Chuan He
出处
期刊:Nature Protocols
[Springer Nature]
日期:2022-01-10
卷期号:17 (2): 402-420
被引量:16
标识
DOI:10.1038/s41596-021-00647-6
摘要
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 N3-kethoxal-assisted labeling. We developed N3-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 N3-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.
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