Psychrophilic proteases dramatically reduce single-cell RNA-seq artifacts: a molecular atlas of kidney development
Psychrophilic proteases dramatically reduce single-cell RNA-seq artifacts: a molecular atlas of kidney development
复制标题
DOI：
10.1242/dev.151142
复制
发表时间：
2017-10-01
期刊：
影响因子：
4.6
通讯作者：
Potter, S. Steven
中科院分区：
文献类型：
--
作者：
Adam, Mike;Potter, Andrew S.;Potter, S. Steven
单细胞 RNA-seq 是一项强大的技术。然而，存在重要的限制，包括将器官或组织分解成单细胞悬浮液的技术挑战。这总是需要在 37 摄氏度下进行酶孵育，预计这会导致基因表达模式的人为变化。在这里，我们描述了一种解离方法，该方法使用从嗜冷微生物中纯化的在寒冷中具有高活性的蛋白酶。整个过程在 6 摄氏度或更低的温度下进行，在这个温度下，哺乳动物的转录机制基本上不活跃，从而有效地“冻结”体内基因表达模式。为了测试这种方法，我们对出生后第 1 天小鼠肾脏的 20,424 个单细胞进行了 RNA 测序，将嗜冷蛋白酶方法的结果与使用 37 摄氏度孵育的程序进行比较。我们证明冷蛋白酶方法可以大大减少基因表达伪影。此外，结果还生成了新生小鼠肾脏的单细胞分辨率基因表达图谱，这是一个有趣的发育时期，此时存在成熟的肾单位，但肾发生仍然极其活跃。
Single-cell RNA-seq is a powerful technique. Nevertheless, there are important limitations, including the technical challenges of breaking down an organ or tissue into a single-cell suspension. Invariably, this has required enzymatic incubation at 37 degrees C, which can be expected to result in artifactual changes in gene expression patterns. Here, we describe a dissociation method that uses a protease with high activity in the cold, purified from a psychrophilic microorganism. The entire procedure is carried out at 6 degrees C or colder, at which temperature mammalian transcriptional machinery is largely inactive, thereby effectively 'freezing in' the in vivo gene expression patterns. To test this method, we carried out RNA-seq on 20,424 single cells from postnatal day 1 mouse kidneys, comparing the results of the psychrophilic protease method with procedures using 37 degrees C incubation. We show that the cold protease method provides a great reduction in gene expression artifacts. In addition, the results produce a single-cell resolution gene expression atlas of the newborn mouse kidney, an interesting time in development when mature nephrons are present yet nephrogenesis remains extremely active.