小柯机器人

美国哈佛大学Beth Stevens小组利用小鼠和人脑的单细胞测序揭示人为和混淆的神经胶质特征。这一研究成果于2022年3月8日在线发表在国际学术期刊《自然—神经科学》上。

研究人员证明了在脑组织上使用酶解会诱发异常的体外基因表达特征，在小胶质细胞中最为突出，这在已发表的文献中很普遍，并会严重干扰下游的分析。为了解决这个问题，研究人员提出了一个经过严格验证的方案，该方案既保留了体内转录谱，又保留了细胞类型的多样性和跨组织类型和物种的产量。研究人员还在死后的人脑单核RNA测序数据集中发现了一个类似的特征，并表明这一特征在新鲜分离的人体组织中通过暴露于体外高温而被诱导出来。总之，这些结果为防止新鲜组织消化过程中人为的基因表达变化提供了一种方法学解决方案，并为今后深入分析死后人类样本中存在的潜在干扰状态提供了参考。

据了解，几乎所有单细胞测序实验的一个关键方面是将完整的组织解离成单细胞悬浮液。虽然许多协议已被优化为最佳细胞产量，但它们往往忽略了解离对体外基因表达的影响。

附：英文原文

Title: Dissection of artifactual and confounding glial signatures by single-cell sequencing of mouse and human brain

Author: Marsh, Samuel E., Walker, Alec J., Kamath, Tushar, Dissing-Olesen, Lasse, Hammond, Timothy R., de Soysa, T. Yvanka, Young, Adam M. H., Murphy, Sarah, Abdulraouf, Abdulraouf, Nadaf, Naeem, Dufort, Connor, Walker, Alicia C., Lucca, Liliana E., Kozareva, Velina, Vanderburg, Charles, Hong, Soyon, Bulstrode, Harry, Hutchinson, Peter J., Gaffney, Daniel J., Hafler, David A., Franklin, Robin J. M., Macosko, Evan Z., Stevens, Beth

Issue&Volume: 2022-03-08

Abstract: A key aspect of nearly all single-cell sequencing experiments is dissociation of intact tissues into single-cell suspensions. While many protocols have been optimized for optimal cell yield, they have often overlooked the effects that dissociation can have on ex vivo gene expression. Here, we demonstrate that use of enzymatic dissociation on brain tissue induces an aberrant ex vivo gene expression signature, most prominently in microglia, which is prevalent in published literature and can substantially confound downstream analyses. To address this issue, we present a rigorously validated protocol that preserves both in vivo transcriptional profiles and cell-type diversity and yield across tissue types and species. We also identify a similar signature in postmortem human brain single-nucleus RNA-sequencing datasets, and show that this signature is induced in freshly isolated human tissue by exposure to elevated temperatures ex vivo. Together, our results provide a methodological solution for preventing artifactual gene expression changes during fresh tissue digestion and a reference for future deeper analysis of the potential confounding states present in postmortem human samples. Marsh et al. demonstrate that enzymatic dissociation induces an aberrant ex vivo gene expression signature, most prominently in microglia, which when not addressed can substantially confound downstream analyses. They also identify a similar signature in postmortem human brain in snRNA-seq.

DOI: 10.1038/s41593-022-01022-8

Source: https://www.nature.com/articles/s41593-022-01022-8

Nature Neuroscience：《自然—神经科学》，创刊于1998年。隶属于施普林格·自然出版集团，最新IF：28.771
官方网址：https://www.nature.com/neuro/
投稿链接：https://mts-nn.nature.com/cgi-bin/main.plex