Differential viral accessibility (DIVA) identifies alterations in chromatin architecture through large-scale mapping of lentiviral integration sites.
Differential viral accessibility (DIVA) identifies alterations in chromatin architecture through large-scale mapping of lentiviral integration sites.
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差异病毒可及性 (DIVA) 通过大规模慢病毒整合位点作图来识别染色质结构的改变。
DOI：
10.1038/s41596-018-0087-5
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发表时间：
2019
期刊：
影响因子：
14.8
通讯作者：
Timms RT
中科院分区：
文献类型：
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作者：
Timms RT
染色质结构的改变在基因表达的表观遗传调控中发挥着重要作用。在这里，我们描述了差异病毒可及性（DIVA）的分步方案，这是一种识别全基因组染色质可及性变化的方法。用于绘制可访问基因组位点的常用方法对检测调控区域中发现的“开放”染色质具有强烈的偏好，但不太适合研究基因体和基因间区域中的染色质可访问性。 DIVA 克服了这一限制，可以查询更广泛的站点。从概念上讲，DIVA 与 ATAC-seq 类似，它依赖于外源 DNA 整合到基因组中来绘制可及染色质图谱，不同之处在于染色质结构是通过绘制外源慢病毒的整合位点来探测的。用慢病毒载体转导一对同基因细胞系，然后进行 PCR 扩增和病毒基因组连接点的 Illumina 测序；所得序列定义了一组独特的慢病毒整合位点，将其进行比较以确定基因组位点在实验细胞和对照细胞之间是否表现出显着改变的可及性。经验丰富的研究人员将花费 6 天的时间来生成慢病毒原液并转导靶细胞，再花 5 天的时间来准备 Illumina 测序文库，并用几个小时来进行生物信息学分析。
Alterations in chromatin structure play a major role in the epigenetic regulation of gene expression. Here, we describe a step-by-step protocol for differential viral accessibility (DIVA), a method for identifying changes in chromatin accessibility genome-wide. Commonly used methods for mapping accessible genomic loci have strong preferences toward detecting ‘open’ chromatin found at regulatory regions but are not well suited to studying chromatin accessibility in gene bodies and intergenic regions. DIVA overcomes this limitation, enabling a broader range of sites to be interrogated. Conceptually, DIVA is similar to ATAC-seq in that it relies on the integration of exogenous DNA into the genome to map accessible chromatin, except that chromatin architecture is probed through mapping integration sites of exogenous lentiviruses. An isogenic pair of cell lines are transduced with a lentiviral vector, followed by PCR amplification and Illumina sequencing of virus–genome junctions; the resulting sequences define a set of unique lentiviral integration sites, which are compared to determine whether genomic loci exhibit significantly altered accessibility between experimental and control cells. Experienced researchers will take 6 d to generate lentiviral stocks and transduce the target cells, a further 5 d to prepare the Illumina sequencing libraries and a few hours to perform the bioinformatic analysis.