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

Abstract

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).

Keywords: Cell Biology; Cell isolation; Flow Cytometry/Mass Cytometry; Neuroscience; RNAseq; Single Cell.

Graphical abstract

Figure 1

Preparation of pasteur pipettes (A) Suspend Pasteur pipette under an oil burner. (B) Allow flame to heat the pipette tip. (C) Measure the internal diameter using a calibration slide under a dissection microscope. Scale bar = 200 μm.

Figure 2

Dissociation set-up Proximity of all equipment is critical to ensure brevity of protocol to maintain health of dissociated neurons. All surfaces should be wiped with 70% ethanol.

Figure 3

Tissue macro dissection (A) 500 μm thick sections of cortex were cut using the adult brain matrix and placed in a petri dish with carbogenated aCSF. (B) One section of cortex was visualized under epifluorescence. The area in the white box was dissected. (C). High magnification of the area in the box from (B), showing individual cell bodies and axonal projections of corticospinal neurons. Scale bar = 2 mm, 500 μm, 50 μm.

Figure 4

Example FACS gating strategy for collecting Rbp4+ neurons from Rbp4 cre mice crossed with tdTomato reporter mice (ai14) 1) Polystyrene beads allow for gating on known size. 2) Based on beads size, size gate of sample is set. 3) Backscatter height and width are used to remove cellular doublets or intact cells plus debris. 4) Non-fluorescent control sample is used to set fluorescence gate (tdTomato from reporter mouse). 5) Collection shows intact cells expressing tdTomato and enriched in cytoplasmic dye.

Figure 5

Neurons following dissociation (A) Corticospinal neurons retrogradely labeled with rAAV-CAG-tdTomato were dissociated according to the protocol above. Robust detection of tdTomato confirms an intact plasma membrane and retained cytoplasm. (B) Enzymatic digestion with 1 mg/mL pronase solution at room temperature without mixing results in a loss of cell integrity and adhesion of non-specific fluorescent debris to nuclei. Neurons were imaged prior to FACS using an Amnis Imagestream. Scale bar = 5 μm.