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

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 nanogramm amounts of moderately degraded RNA enabled us to detect ∼15,000 different transcripts in fluorescently-labeled cholinergic neuron populations. The versatile LCM-Seq method showed excellent accuracy in quantitative studies, with 2,891 transcripts expressed differentially between the spatially defined and clinically relevant cholinergic neuron populations of the caudate-putamen and medial septum.

Estrogen-dependent development and transcriptome regulation of the lateral septal kisspeptin system

“…A laser capture microdissection/RNA-sequencing ('LCM-Seq') method developed in our laboratory (42) has enabled us to study and identify the estrogen-regulated transcripts of KP ARC (43) and KP RP3V (25) neurons. Here we characterized the transcriptome landscape of KP LS neurons using the same animal models and protocols to allow the comparative analysis of the different KP cell types.…”

“…The LS was sectioned serially with a cryostat and ZsGreen-tagged KP neurons were isolated with LCM (25,43). As the low number of KP LS cells in individual mice may result in suboptimal detection sensitivity and quantification precision/accuracy of the LCM-Seq protocol (42), ~360 fluorescently-tagged neurons were pooled proportionally from 3 brains to prepare each RNA sample and cDNA library, followed by Illumina sequencing (25,43). Meta-analysis of the two hypothalamic KP neuron transcriptomes was carried out from open-access public repositories associated with the original publications (25,43).…”

“…While much attention has been paid to the reproductive significance of the two hypothalamic KP neuron populations, functions of the extra-hypothalamic KP neurons are still relatively unexplored. Here we have used an LCM-Seq method developed in our laboratory (42) to study the transcriptome profile of KP LS neurons and its estrogen-dependent regulation. This method allowed us to identify more than 15,000 transcripts which could then be compared to the transcripts of KP ARC and KP RP3V neurons we studied earlier using the same methodologies and animal models (25,43).…”