SPRITE: a genome-wide method for mapping higher-order 3D interactions in the nucleus using combinatorial split-and-pool barcoding.
SPRITE: a genome-wide method for mapping higher-order 3D interactions in the nucleus using combinatorial split-and-pool barcoding.
复制标题
SPRITE：一种全基因组方法，使用组合分割和池条形码来绘制细胞核中高阶 3D 相互作用。
DOI：
10.1038/s41596-021-00633-y
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发表时间：
2022
期刊：
影响因子：
14.8
通讯作者：
Guttman,Mitchell
中科院分区：
文献类型：
--
作者：
Quinodoz,SofiaA;Bhat,Prashant;Chovanec,Peter;Jachowicz,JoannaW;Ollikainen,Noah;Detmar,Elizabeth;Soehalim,Elizabeth;Guttman,Mitchell
基因调控中的一个基本问题是细胞类型特异性基因表达如何受到每个细胞核内DNA包装的影响。我们最近开发了分裂池识别的相互作用的标签扩展（SPRITE），这使得映射的高阶相互作用的核内。SPRITE通过交联相互作用的DNA，RNA和蛋白质分子，然后通过迭代的分裂和合并条形码方法绘制DNA-DNA空间排列。交联复合物内的所有DNA分子通过在96孔板上重复分裂复合物，将分子与独特的标签序列连接，并在重复标记之前将所有复合物汇集到单个孔中来条形码化。由于交联复合物中的所有分子都是共价连接的，因此它们将在每一轮分裂和合并过程中分类在一起，并将获得相同系列的SPRITE标签，我们将其称为条形码。对DNA片段及其相关条形码进行测序，并匹配共享相同条形码的所有读段以重建相互作用。SPRITE精确地映射了细胞核内的成对DNA相互作用，并测量了数千个同时相互作用的分子之间发生的高阶空间接触。在这里，我们提供了SPRITE实验步骤的详细协议，包括视频（https：//youtu.be/6SdWkBxQGlg）。此外，我们在GitHub上提供了一个自动计算管道，允许实验者从原始fastq文件开始无缝生成SPRITE交互矩阵。该方案从细胞交联到高通量测序的实验步骤需要约5 d，数据处理需要1 d。
A fundamental question in gene regulation is how cell-type-specific gene expression is influenced by the packaging of DNA within the nucleus of each cell. We recently developed Split-Pool Recognition of Interactions by Tag Extension (SPRITE), which enables mapping of higher-order interactions within the nucleus. SPRITE works by cross-linking interacting DNA, RNA and protein molecules and then mapping DNA–DNA spatial arrangements through an iterative split-and-pool barcoding method. All DNA molecules within a cross-linked complex are barcoded by repeatedly splitting complexes across a 96-well plate, ligating molecules with a unique tag sequence, and pooling all complexes into a single well before repeating the tagging. Because all molecules in a cross-linked complex are covalently attached, they will sort together throughout each round of split-and-pool and will obtain the same series of SPRITE tags, which we refer to as a barcode. The DNA fragments and their associated barcodes are sequenced, and all reads sharing identical barcodes are matched to reconstruct interactions. SPRITE accurately maps pairwise DNA interactions within the nucleus and measures higher-order spatial contacts occurring among up to thousands of simultaneously interacting molecules. Here, we provide a detailed protocol for the experimental steps of SPRITE, including a video (https://youtu.be/6SdWkBxQGlg). Furthermore, we provide an automated computational pipeline available on GitHub that allows experimenters to seamlessly generate SPRITE interaction matrices starting with raw fastq files. The protocol takes ~5 d from cell cross-linking to high-throughput sequencing for the experimental steps and 1 d for data processing.