Development of a versatile LCM-Seq method for spatial transcriptomics of fluorescently-tagged cholinergic neuron populations

Rumpler, Éva and Göcz, Balázs Gergő and Skrapits, Katalin and Sárvári, Miklós and Takács, Szabolcs Ferenc and Farkas, Imre and Solymosi, Norbert and Hrabovszky, Erik (2023) Development of a versatile LCM-Seq method for spatial transcriptomics of fluorescently-tagged cholinergic neuron populations. JOURNAL OF BIOLOGICAL CHEMISTRY, 299 (9). ISSN 0021-9258

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Abstract

Single-cell transcriptomics are powerful tools to define neuronal cell types based on co-expressed gene clusters. Limited RNA input in these technologies necessarily compromises transcriptome coverage and accuracy of differential expression analysis. We propose that bulk RNA-sequencing of neuronal pools defined by spatial position offers an alternative strategy to overcome these technical limitations. We report an LCM-Seq method which allows deep transcriptome profiling of fluorescently-tagged neuron populations isolated with laser-capture microdissection (LCM) from histological sections of transgenic mice. Mild formaldehyde-fixation of ZsGreen marker protein, LCM sampling of ∼300 pooled neurons, followed by RNA isolation, library preparation and RNA-sequencing with methods optimized for nanogram amounts of moderately degraded RNA enabled us to detect ∼15,000 different transcripts in fluorescently-labeled cholinergic neuron populations. The LCM-Seq approach showed excellent accuracy in quantitative studies, allowing us to detect 2,891 transcripts expressed differentially between the spatially defined and clinically relevant cholinergic neuron populations of the dorsal caudate-putamen and medial septum. In summary, the LCM-Seq method we report in this study is a versatile, sensitive and accurate bulk sequencing approach to study the transcriptome profile and differential gene expression of fluorescently tagged neuronal populations isolated from transgenic mice with high spatial precision.

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