μDamID: A Microfluidic Approach for Joint Imaging and Sequencing of Protein-DNA Interactions in Single Cells.
μDamID: A Microfluidic Approach for Joint Imaging and Sequencing of Protein-DNA Interactions in Single Cells.
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μDamid：一种微流体方法，用于单个细胞中蛋白DNA相互作用的关节成像和测序。
DOI：
10.1016/j.cels.2020.08.015
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发表时间：
2020-10-21
期刊：
影响因子：
9.3
通讯作者：
Streets A
中科院分区：
文献类型：
--
作者：
Altemose N;Maslan A;Rios-Martinez C;Lai A;White JA;Streets A
DNA腺嘌呤甲基转移酶鉴定(DamID)通过甲基化每个蛋白质-DNA相互作用位点附近的腺嘌呤碱基，然后选择性地扩增和测序这些甲基化区域，来测量蛋白质的DNA结合史。此外，这些相互作用可以使用m6A-Tracer可视化，m6A-Tracer是一种与甲基腺嘌呤结合的荧光蛋白。在这里，我们将这些成像和测序技术结合在一个集成的微流控平台(μDAMID)中，该平台可以实现单细胞分离、成像和分选，然后是DAMID。我们使用μDAMID和一种改进的m6A-Tracer蛋白来生成来自单个人类细胞的配对成像和测序数据。我们验证了Lamin-B1蛋白和LADs之间的相互作用，观察了可变的3D染色质组织和广泛的基因调控模式，并联合测量了Dam表达和背景甲基化的单细胞异质性。μDAMID提供了独特的能力，可以比较每个细胞和细胞之间的配对成像和测序数据，从而能够联合分析核定位、序列同一性和蛋白质-DNA相互作用的可变性。补充资料中包含了本文件透明的同行审查过程的记录。每个细胞的行为在很大程度上取决于调节基因表达的蛋白质-DNA相互作用。在这里，作者设计并建造了一种微流控设备，使用户能够分类和分离活的单细胞，以高分辨率成像特定蛋白质-DNA相互作用的空间位置，然后从这些相互作用部位扩增DNA并进行测序。这首次提供了配对的成像和测序数据，揭示了单个细胞内跨基因组的蛋白质-DNA相互作用的空间位置和序列同一性。
DNA adenine methyltransferase identification (DamID) measures a protein’s DNA-binding history by methylating adenine bases near each protein-DNA interaction site and then selectively amplifying and sequencing these methylated regions. Additionally, these interactions can be visualized using m6A-Tracer, a fluorescent protein that binds to methyladenines. Here, we combine these imaging and sequencing technologies in an integrated microfluidic platform (μDamID) that enables single-cell isolation, imaging, and sorting, followed by DamID. We use μDamID and an improved m6A-Tracer protein to generate paired imaging and sequencing data from individual human cells. We validate interactions between Lamin-B1 protein and lamina-associated domains (LADs), observe variable 3D chromatin organization and broad gene regulation patterns, and jointly measure single-cell heterogeneity in Dam expression and background methylation. μDamID provides the unique ability to compare paired imaging and sequencing data for each cell and between cells, enabling the joint analysis of the nuclear localization, sequence identity, and variability of protein-DNA interactions. A record of this paper’s transparent peer review process is included in the Supplemental Information. Each cell’s behavior depends largely on protein-DNA interactions that regulate gene expression. Here, the authors designed and built a microfluidic device enabling the user to sort and isolate live single cells, image the spatial location of specific protein-DNA interactions at high resolution, and then amplify and sequence DNA from these interaction sites. This provides, for the first time, paired imaging and sequencing data that reveal both the spatial location and sequence identity of protein-DNA interactions across the genome within single cells.
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影响因子：
7
作者：
Lenain C;de Graaf CA;Pagie L;Visser NL;de Haas M;de Vries SS;Peric-Hupkes D;van Steensel B;Peeper DS
通讯作者：
Peeper DS
影响因子：
16.6
作者：
Kim S;De Jonghe J;Kulesa AB;Feldman D;Vatanen T;Bhattacharyya RP;Berdy B;Gomez J;Nolan J;Epstein S;Blainey PC
通讯作者：
Blainey PC
影响因子：
64.5
作者：
Kind J;Pagie L;de Vries SS;Nahidiazar L;Dey SS;Bienko M;Zhan Y;Lajoie B;de Graaf CA;Amendola M;Fudenberg G;Imakaev M;Mirny LA;Jalink K;Dekker J;van Oudenaarden A;van Steensel B
通讯作者：
van Steensel B
影响因子：
14.9
作者：
Bernhofer M;Goldberg T;Wolf S;Ahmed M;Zaugg J;Boden M;Rost B
通讯作者：
Rost B
影响因子：
30.8
作者：
Grosselin, Kevin;Durand, Adeline;Gerard, Annabelle
通讯作者：
Gerard, Annabelle