ATAC-Seq-based Identification of Extrachromosomal Circular DNA in Mammalian Cells and Its Validation Using Inverse PCR and FISH
ATAC-Seq-based Identification of Extrachromosomal Circular DNA in Mammalian Cells and Its Validation Using Inverse PCR and FISH
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DOI：
10.21769/bioprotoc.4003
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发表时间：
2021-05-05
期刊：
影响因子：
0.8
通讯作者：
Dutta, Anindya
中科院分区：
文献类型：
--
作者：
Su, Zhangli;Saha, Shekhar;Dutta, Anindya
包括我们在内的多个实验室最近的研究证明了从酵母到人类的染色体外环DNA(EccDNA)的重要性(Shibata等人，2012；Dillon等人，2015；Moller等人，2016；Kumar等人，2017；Turner等人，2017；Kim等人，2020)。最近，人们发现癌细胞通过放大eccDNA上的癌基因获得了一种选择性优势，这导致了基因组的不稳定性(Wu等人，2019；Kim等人，2020)。以前，我们已经纯化了环状DNA，并使用滚环扩增和高通量测序的方法丰富了群体，以基于唯一的连接序列来鉴定eccDNA。然而，滚动圆扩增的eccDNA鉴定方法偏向于小圆。在这里，我们报告了一种滚动圈无关的方法来检测人类癌细胞中的eccDNA。我们展示了一种敏感而稳健的循序渐进的工作流程来寻找新的eccDNA，使用ATAC-seq(使用测序对转座酶可访问的染色质进行分析)与Circle_finder生物信息学算法相结合来预测eccDNA，然后使用两种独立的方法-反向PCR和中期FISH(荧光原位杂交)进行验证。
Recent studies from multiple labs including ours have demonstrated the importance of extrachromosomal circular DNA (eccDNA) from yeast to humans (Shibata et al., 2012; Dillon et al., 2015; Moller et al., 2016; Kumar et al., 2017; Turner et al., 2017; Kim et al., 2020). More recently, it has been found that cancer cells obtain a selective advantage by amplifying oncogenes on eccDNA, which drives genomic instability (Wu et al., 2019; Kim et al, 2020). Previously, we have purified circular DNA and enriched the population using rolling circle amplification followed by high-throughput sequencing for the identification of eccDNA based on the unique junctional sequence. However, eccDNA identification by rolling circle amplification is biased toward small circles. Here, we report a rolling circle-independent method to detect eccDNA in human cancer cells. We demonstrate a sensitive and robust step-by-step workflow for finding novel eccDNAs using ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) combined with a Circle_finder bioinformatics algorithm to predict the eccDNAs, followed by its validation using two independent methods, inverse PCR and metaphase FISH (Fluorescence in situ Hybridization).