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Preparation of Cells from Embryonic Organs for Single-Cell RNA Sequencing

机译：单细胞RNA测序的胚胎器官的制备

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摘要

Although single-cell RNA sequencing (scRNA-seq) has become one of the most powerful methods available for transcriptome analysis, the quality of scRNA-seq data largely depends on cell preparation. Cell preparation from cultured cells and tissues requires different methods because of the inherent differences between these two categories of cells. Compared to cultured cells, tissues have more extracellular matrix, and the cells are generally more adherent and thus difficult to dissociate. The challengeis to achieve sufficient dissociation, cell counts, and viability all at the same time. This protocol describes approaches that help achieve these goals. These include a cold dissociation technique using cryophilic proteases active at cold temperature,timing of trituration during protease digestion, as well as filtration and washing methods that optimize cell viability and retention. Materials and equipment that optimize the process also discussed.
机译：虽然单细胞RNA测序（ScRNA-SEQ）已成为可用于转录组分析的最强大的方法之一，但SCRNA-SEQ数据的质量在很大程度上取决于细胞制备。 由于这两类细胞之间的固有差异，来自培养细胞和组织的细胞制剂需要不同的方法。 与培养的细胞相比，组织具有更多细胞外基质，并且细胞通常更粘附，因此难以解离。 挑战在同时实现足够的解离，细胞计数和可行性。 本协议描述了有助于实现这些目标的方法。 这些包括在寒冷温度下活跃，蛋白酶消化期间氚化的时间，以及优化细胞活力和保留的过滤和洗涤方法的冷冻蛋白酶的冷解离方法。 还讨论了优化该过程的材料和设备。

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