Extended Data Fig. 4: Isolation of C. elegans touch receptor neurons by the dissociation-FACS method. | Nature Methods

Extended Data Fig. 4: Isolation of C. elegans touch receptor neurons by the dissociation-FACS method.

From: Femtosecond laser microdissection for isolation of regenerating C. elegans neurons for single-cell RNA sequencing

Extended Data Fig. 4

a-d, FACSAria II gate settings for isolating living GFP-labeled C. elegans touch receptor neurons from dissociated animals. Prior to FACS, the cell suspension was filtered through a 5 μm syringe filter to prevent clogging. SSC-A, SSC-W, and FSC gates were set up to exclude debris and clusters of cells. We stained the dead neurons with propidium iodide. GFP + PI- events were sorted into 96 well plates or 1.5 mL conical tubes containing lysis buffer. The GFP threshold was determined in reference to the autofluorescence of cells from dissociated N2 worms. The PI threshold was determined by test sorting a small number of PI-stained cells and observing two distinct populations of live/dead cells. e, Histogram of GFP levels of the isolated neurons. f, Cell suspension prior to sorting and g, example of a collected neuron. Green: GFP. Red: PI. Multiple locations were imaged to confirm GFP + neurons using the fluorescent microscope (confirmed with 3 independent sample preparation). Scale bar: 5 μm. h, Quantification of cDNA library prepared from one of the GFP + sorted cells using FACS method.

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