Systematic assessment of tissue dissociation and storage biases in single-cell and single-nucleus RNA-seq workflows
Systematic assessment of tissue dissociation and storage biases in single-cell and single-nucleus RNA-seq workflows
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DOI：
10.1186/s13059-020-02048-6
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发表时间：
2020-06-02
期刊：
影响因子：
12.3
通讯作者：
Forrest, Alistair R. R.
中科院分区：
文献类型：
--
作者：
Denisenko, Elena;Guo, Belinda B.;Forrest, Alistair R. R.
背景单细胞RNA测序已被广泛用于估计异质组织的细胞组成和获得单个细胞的转录图谱。已经提出了多种方法来优化单细胞的样品解离和存储，单核分析方法也是如此。缺乏的是对它们的相对偏向和利益进行系统的比较。结果在这里，我们使用两种组织分离方案比较了成年小鼠肾脏制备的单细胞悬液的基因表达和细胞组成。对于每个样本，我们还将新鲜细胞与冷冻保存和甲醇固定的细胞进行比较。最后，我们将这些单细胞数据与使用三个单核RNA测序工作流程产生的数据进行比较。我们的数据证实了先前的报告，即在冰上消化可以避免在37摄氏度解离时观察到的压力反应。它还揭示了更丰富的细胞类型，无论是在冷的还是暖的分离中，这可能代表了需要更温和或更苛刻的条件才能完整释放的种群。对于细胞储存，分离细胞的超低温保存会导致上皮细胞类型的重大损失；相比之下，甲醇固定保持了细胞组成，但会导致环境中的RNA泄漏。最后，观察到单细胞和单核RNA测序文库之间的细胞类型组成差异。特别是，我们注意到T、B和NK淋巴细胞在单核文库中的表达不足。结论对回收的细胞类型及其在整个工作流程中的转录谱进行了系统的比较，突出了特定于方案的偏见，从而使研究人员能够开始单细胞实验以做出明智的选择。
Background Single-cell RNA sequencing has been widely adopted to estimate the cellular composition of heterogeneous tissues and obtain transcriptional profiles of individual cells. Multiple approaches for optimal sample dissociation and storage of single cells have been proposed as have single-nuclei profiling methods. What has been lacking is a systematic comparison of their relative biases and benefits. Results Here, we compare gene expression and cellular composition of single-cell suspensions prepared from adult mouse kidney using two tissue dissociation protocols. For each sample, we also compare fresh cells to cryopreserved and methanol-fixed cells. Lastly, we compare this single-cell data to that generated using three single-nucleus RNA sequencing workflows. Our data confirms prior reports that digestion on ice avoids the stress response observed with 37 degrees C dissociation. It also reveals cell types more abundant either in the cold or warm dissociations that may represent populations that require gentler or harsher conditions to be released intact. For cell storage, cryopreservation of dissociated cells results in a major loss of epithelial cell types; in contrast, methanol fixation maintains the cellular composition but suffers from ambient RNA leakage. Finally, cell type composition differences are observed between single-cell and single-nucleus RNA sequencing libraries. In particular, we note an underrepresentation of T, B, and NK lymphocytes in the single-nucleus libraries. Conclusions Systematic comparison of recovered cell types and their transcriptional profiles across the workflows has highlighted protocol-specific biases and thus enables researchers starting single-cell experiments to make an informed choice.