Mazelis Ignas, Sun Haoxiang, Kulkarni Arpita, Torre Theresa, Klein Allon M
Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA.
Single Cell Core, Harvard Medical School, Boston, MA 02115, USA.
bioRxiv. 2025 Mar 17:2025.03.14.642839. doi: 10.1101/2025.03.14.642839.
Single-cell genomics encompasses a set of methods whereby hundreds to millions of cells are individually subjected to multiplexed assays including sequencing DNA, chromatin accessibility or modification, RNA, or combinations thereof. These methods enable unbiased, systematic discovery of cellular phenotypes and their dynamics. Many functional genomic methods, however, require multiple steps that cannot be easily scaled to high throughput, including assays on living cells. Here we develop capsules with amphiphilic gel envelopes (CAGEs), which selectively retain cells, mRNA, and gDNA, while allowing free diffusion of media, enzymes and reagents. CAGEs enable carrying out high-throughput assays that require multiple steps, including combining genomics with live-cell assays. We establish methods for barcoding CAGE DNA and RNA libraries, and apply them to measure persistence of gene expression programs by capturing the transcriptomes of tens of thousands of expanding clones in CAGEs. The compatibility of CAGEs with diverse enzymatic reactions will facilitate the expansion of the current repertoire of single-cell, high-throughput measurements and extend them to live-cell assays.
单细胞基因组学涵盖了一系列方法，通过这些方法，数以百计到数百万计的细胞被单独进行多重分析，包括对DNA、染色质可及性或修饰、RNA或它们的组合进行测序。这些方法能够无偏见、系统地发现细胞表型及其动态变化。然而，许多功能基因组学方法需要多个不易扩展到高通量的步骤，包括对活细胞的分析。在这里，我们开发了具有两亲性凝胶包膜的胶囊（CAGEs），它能选择性地保留细胞、mRNA和基因组DNA，同时允许培养基、酶和试剂自由扩散。CAGEs能够进行需要多个步骤的高通量分析，包括将基因组学与活细胞分析相结合。我们建立了对CAGE DNA和RNA文库进行条形码标记的方法，并将其应用于通过捕获CAGEs中数万个扩增克隆的转录组来测量基因表达程序的持久性。CAGEs与多种酶促反应的兼容性将有助于扩展当前单细胞高通量测量的方法，并将其扩展到活细胞分析中。
