Deep sequencing reveals cell-type specific patterns of single cell transcriptome variation. Deep sequencing reveals cell-type specific patterns of single cell transcriptome variation

We present high quality deep read-depth single cell RNA sequencing for 91 cells from five mouse tissues and 18 cells from two rat tissues, along with 30 control samples of bulk RNA diluted to single-cell levels. We find that transcriptomes differ globally across tissues with regard to the number of genes expressed, the average expression patterns, and within cell-type variation patterns. We develop methods to filter genes for reliable quantification and to calibrate biological variation. All cell types include genes with high variability in expression, in a tissue-specific manner. We also find evidence that single cell variability of neuronal genes in mice is correlated with that in rats consistent with the hypothesis that levels of variation may be conserved. Overall design: From an initial 143 cells we identified 107 high quality samples with deep genic coverage, including 13 brown adipocytes, 19 cardiomyocytes, 19 cortical pyramidal neurons and 18 hippocampal pyramidal neurons from embryonic mouse, 8 cortical pyramidal neurons and 8 hippocampal pyramidal neurons from embryonic rat, and 22 serotonergic neurons from adult mouse. While mouse data is collected from three strains of mice at different ages, each cell type dataset is internally consistent. There are no technical replicates of single-cell samples. We additionally prepared 30 control samples, amplifications of bulk total cardiomyocyte RNA diluted to single cell quantities.

本研究提供了高质量的高测序深度单细胞RNA测序（single cell RNA sequencing）数据，涵盖91个来自5种小鼠组织的细胞、18个来自2种大鼠组织的细胞，以及30份经稀释至单细胞水平的bulk RNA对照样本。研究发现，不同组织的转录组在表达基因数量、平均表达模式以及细胞类型内变异模式等维度均存在全局性差异。本研究开发了用于基因可靠定量的筛选方法，以及生物变异校准方法。所有细胞类型中均存在表达变异程度较高的基因，且该特征呈现组织特异性。本研究还发现证据表明，小鼠神经元基因的单细胞变异程度与大鼠神经元基因的单细胞变异程度存在相关性，这与"变异水平可能具有保守性"的假说相符。
实验整体设计：初始的143个细胞中，我们筛选得到107份具有深度基因覆盖度的高质量样本，其中包括：13个胚胎小鼠棕色脂肪细胞（brown adipocytes）、19个胚胎小鼠心肌细胞（cardiomyocytes）、19个胚胎小鼠大脑皮层锥体神经元（cortical pyramidal neurons）、18个胚胎小鼠海马锥体神经元（hippocampal pyramidal neurons）、8个胚胎大鼠大脑皮层锥体神经元（cortical pyramidal neurons）、8个胚胎大鼠海马锥体神经元（hippocampal pyramidal neurons），以及22个成年小鼠5-羟色胺能神经元（serotonergic neurons）。尽管小鼠数据来自3个不同品系、不同年龄段的小鼠，但每种细胞类型的数据集内部均具有一致性。本研究未设置单细胞样本的技术重复。此外，本研究还制备了30份对照样本：将bulk总心肌细胞RNA稀释至单细胞量级后进行扩增得到的样本。