Effective microtissue RNA extraction coupled with Smart-seq2 for reproducible and robust spatial transcriptome analysis
Effective microtissue RNA extraction coupled with Smart-seq2 for reproducible and robust spatial transcriptome analysis
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DOI：
10.1038/s41598-020-63495-6
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发表时间：
2020-04-27
影响因子：
4.6
通讯作者：
Takeyama, Haruko
中科院分区：
文献类型：
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作者：
Yamazaki, Miki;Hosokawa, Masahito;Takeyama, Haruko
空间转录组学有助于理解组织的分子组织，并提供在形态学背景下对细胞功能的见解。为了在空间转录组学中获得可重复的结果，我们必须在图像采集和生物分子收集过程中保持组织形态和RNA分子的稳定性。在这里，我们开发了一种组织处理方法，用于从组织显微切割样品中进行稳健且可重复的RNA-seq。在这种方法中，我们抑制RNA降解新鲜冷冻组织标本的脱水固定和有效地收集了少量的RNA分子从显微切割样品的磁珠。我们展示了小鼠肝脏和大脑的空间转录组分析系列显微切割样品（直径100 μ m，厚度10 μ m）由显微切割打孔系统。使用我们的方法，我们可以防止RNA在室温下降解，并有效地产生Smart-seq 2测序文库。这导致在外显子区域中的可再现的序列读取作图，并且与RNA-seq分析中的非固定样品相比，检测到超过2000个基因。我们的方法将适用于各种转录组分析，为组织标本中区域特异性基因表达提供信息。
Spatial transcriptomics is useful for understanding the molecular organization of a tissue and providing insights into cellular function in a morphological context. In order to obtain reproducible results in spatial transcriptomics, we have to maintain tissue morphology and RNA molecule stability during the image acquisition and biomolecule collection processes. Here, we developed a tissue processing method for robust and reproducible RNA-seq from tissue microdissection samples. In this method, we suppressed RNA degradation in fresh-frozen tissue specimens by dehydration fixation and effectively collected a small amount of RNA molecules from microdissection samples by magnetic beads. We demonstrated the spatial transcriptome analysis of the mouse liver and brain in serial microdissection samples (100 mu m in a diameter and 10 mu m in thickness) produced by a microdissection punching system. Using our method, we could prevent RNA degradation at room temperature and effectively produce a sequencing library with Smart-seq2. This resulted in reproducible sequence read mapping in exon regions and the detection of more than 2000 genes compared to non-fixed samples in the RNA-seq analysis. Our method would be applied to various transcriptome analyses, providing the information for region specific gene expression in tissue specimens.