The functioning of tissues is fundamentally dependent upon not only the phenotypes of the constituent cells but also their spatial organization in the tissue. However, acquisition of comprehensive transcriptomes of spatially- and phenotypically-defined cells in situ remains challenging. Here we present a general and robust method based on immunofluorescence-guided laser capture microdissection (immuno-LCM-RNAseq) to acquire finely resolved spatial transcriptomes including isoforms with as few as tens of cells from snap-frozen or RNAlater-treated clinical tissues, circumventing the problem of significant RNA degradation during this time-consuming process. The efficacy of this approach is exemplified by the characterization of differences at the transcript isoform level between the mouse small intestine lacteal cells at the tip versus the main capillary body. With the extensive repertoire of specific antibodies that are presently available, our method provides a powerful means by which spatially resolved cellular states can now be delineated in situ with preserved tissues. Moreover, such high quality spatial transcriptomes defined by immunomarkers can be used to compare with the phenotypes derived from single-cell RNAseq of dissociated cells as well as applied to bead-based spatial transcriptomic approaches that require such information a priori for cell identification.

中文翻译：

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使用基于免疫荧光的激光捕获显微切割从保存组织中稳健获取高分辨率空间转录组

组织的功能从根本上不仅取决于组成细胞的表型，还取决于它们在组织中的空间组织。然而，原位获得空间和表型定义的细胞的综合转录组仍然具有挑战性。在这里，我们提出了一种基于免疫荧光引导的激光捕获显微切割 (immuno-LCM-RNAseq) 的通用且稳健的方法，以获取精细解析的空间转录组，包括来自速冻或 RNAlater 处理的临床组织中少至数十个细胞的同种型，规避在这个耗时的过程中 RNA 显着降解的问题。这种方法的功效通过表征小鼠小肠乳头细胞与主要毛细管体之间转录亚型水平的差异来体现。凭借目前可用的大量特异性抗体，我们的方法提供了一种强大的手段，现在可以原位描绘空间分辨的细胞状态用保存的组织。此外，这种由免疫标记物定义的高质量空间转录组可用于与来自分离细胞的单细胞 RNAseq 的表型进行比较，以及应用于需要此类先验信息进行细胞识别的基于珠子的空间转录组学方法。