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 °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.

中文翻译：

---

单细胞和单核 RNA-seq 工作流程中组织解离和储存偏差的系统评估

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并获得单个细胞的转录谱。已经提出了用于最佳样品解离和单细胞存储的多种方法，以及单核分析方法。一直缺乏的是对它们的相对偏见和收益的系统比较。结果 在这里，我们比较了使用两种组织解​​离方案从成年小鼠肾脏制备的单细胞悬液的基因表达和细胞组成。对于每个样品，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，我们将此单细胞数据与使用三个单核 RNA 测序工作流程生成的数据进行比较。我们的数据证实了先前的报告，即在冰上消化避免了在 37°C 解离时观察到的应激反应。它还揭示了在冷或暖解离中更丰富的细胞类型，这可能代表需要更温和或更苛刻条件才能完整释放的细胞群。对于细胞储存，解离细胞的冷冻保存会导致上皮细胞类型的大量丢失；相比之下，甲醇固定保持细胞组成，但遭受环境 RNA 泄漏。最后，在单细胞和单核 RNA 测序文库之间观察到细胞类型组成差异。特别是，我们注意到单核文库中 T、B 和 NK 淋巴细胞的代表性不足。