Data_Sheet_6_Protecting RNA quality for spatial transcriptomics while improving immunofluorescent staining quality.PDF

In comparison to bulk sequencing or single cell sequencing, spatial transcriptomics preserves the spatial information in tissue slices and can even be mapped to immunofluorescent stainings, allowing translation of gene expression information into their spatial context. This enables to unravel complex interactions of neighboring cells or to link cell morphology to transcriptome data. The 10× Genomics Visium platform offers to combine spatial transcriptomics with immunofluorescent staining of cryo-sectioned tissue slices. We applied this technique to fresh frozen mouse brain slices and developed a protocol that still protects RNA quality while improving buffers for immunofluorescent staining. We investigated the impact of various parameters, including fixation time and buffer composition, on RNA quality and antibody binding. Here, we propose an improved version of the manufacturer protocol, which does not alter RNA quality and facilitates the use of multiple additional antibodies that were not compatible with the manufacturer protocol before. Finally, we discuss the influence of various staining parameters, which contribute to the development of application specific staining protocols.

与批量测序（bulk sequencing）及单细胞测序相比，空间转录组学（spatial transcriptomics）可保留组织切片内的空间位置信息，还能与免疫荧光染色（immunofluorescent staining）成像结果进行空间配准，从而将基因表达信息与其所处的空间背景进行整合。这一特性可助力解析相邻细胞间的复杂相互作用，或是将细胞形态与转录组（transcriptome）数据进行关联。10× Genomics公司的Visium平台可实现空间转录组学与冰冻切片组织免疫荧光染色的联合检测。我们将该技术应用于新鲜冷冻的小鼠脑组织切片，并开发出一套既能保障RNA质量，又能优化免疫荧光染色缓冲液的实验方案。我们探究了包括固定时长、缓冲液配方在内的多项参数对RNA质量与抗体结合效果的影响。在此，我们提出了厂商原始实验方案的优化版本：该优化方案不会对RNA质量造成影响，同时可兼容此前厂商方案无法适配的多种额外抗体。最后，我们探讨了多项染色参数的影响，这将为适配特定应用场景的染色方案开发提供参考。