Dissociation of intact adult mouse cortical projection neurons for single-cell RNA-seq

Abstract

Summary This protocol provides an improved pipeline for dissociating intact projection neurons from adult mouse cortex for applications including droplet and plate-based single-cell RNA sequencing, qPCR, immunocytochemistry, and long-term in vitro cell culture. This protocol provides a robust and reproducible dissociation pipeline that uses exclusively off-the-shelf reagents, not requiring the use of expensive dissociation kits. The unique incubation steps, in combination with the FACS gating strategy, results in unparalleled enrichment for intact cortical neurons from the adult brain. For complete details on the use and execution of this protocol, please refer to Golan et al. (2021) .

Protocol for differential multi-omic analyses of distinct cell types in the mouse cerebral cortex

“…Lymphocytes and suspension cells are usually run through a 70 μm nozzle, while larger cells, such as adherent cell lines and dissociated primary cells are sorted with a 100 μm nozzle. Dissociated cortical neurons however are rather small in diameter: Pyramidal neurons have reportedly a soma diameter of 10–20 μm 85 , 86 while all processes, dendrites and the axon are collapsed and retracted during dissociation. Note: Weigh the advantage of a larger nozzle size against a decreased flow rate that results in a prolonged sorting time that can ultimately lead to apoptosis and a reduced quality of the isolated proteins.…”

“… 1 , 2 , 3 , 91 , 92 , 93 On the other hand, it has proven difficult to isolate intact adult corticospinal neurons from mice at postnatal age – while maintaining cytoplasmic integrity for comprehensive mRNA sequence analysis – as their large projection axon was reportedly lost upon dissociation and neuron fragility was related to axon length. 85 Furthermore, the dissociation of primary neurons can cause neuron death, which often interferes with transcriptome or epigenome analysis, e.g., ATAC-sequencing protocols. 94 , 95 Nevertheless, this protocol has been successfully applied for differential quantitative mass spectrometric proteomic analysis of migrating cortical neurons.…”

“…Lymphocytes and suspension cells are usually run through a 70 μm nozzle, while larger cells, such as adherent cell lines and dissociated primary cells are sorted with a 100 μm nozzle. Dissociated cortical neurons however are rather small in diameter: Pyramidal neurons have reportedly a soma diameter of 10–20 μm 85 , 86 while all processes, dendrites and the axon are collapsed and retracted during dissociation.…”

Microfluidics for Neuronal Cell and Circuit Engineering

“…In this regard, integration of in vivo or in vitro electrophysiological recordings and morphological evaluations combined with scRNA-Seq data of the same cells provides information to precisely map neuronal subtypes and predict their functional contributions in brain networks. ,− Patch-Seq is an example of a low-throughput method capable of linking the transcriptomic profile of neuronal cells to their neurophysiological and morphological phenotypes and can also be used to investigate the cellular response to diverse chemical stimuli. − Notably, while high-throughput automated patch-clamp electrophysiology tools are available since the 1990s and early 2000s, they still need to be integrated with scRNA-Seq platforms . However, major challenges remain: that dissociated neuronal cells commonly used in scRNA-Seq experiments are not compatible with patch-clamp recordings because they often lose their dendrites and axons in the dissociation process. , Therefore, a key point that needs to be considered in designing the next generation of microfluidic screening platforms is the feasibility of integrating molecular profiling with functional and morphological phenotyping approaches to achieve high-throughput multimodal single-cell profiling platforms. Another challenge lies on the difficulty to capture the dynamic transcriptional states of neurons as they differentiate from stem cells with full functional and morphological features, as current technologies are limited to capturing snapshots of these characteristics at specific time points. , …”

“…216 because they often lose their dendrites and axons in the dissociation process. 217,218 Therefore, a key point that needs to be considered in designing the next generation of microfluidic screening platforms is the feasibility of integrating molecular profiling with functional and morphological phenotyping approaches to achieve high-throughput multimodal single-cell profiling platforms. Another challenge lies on the difficulty to capture the dynamic transcriptional states of neurons as they differentiate from stem cells with full functional and morphological features, as current technologies are limited to capturing snapshots of these characteristics at specific time points.…”