Summary
This dataset contains 67,346 cells (36,117 neurons) collected from 6 samples of day 1 him-8(e1498) adults.
Data preparation
him-8(e1498) cultures contain approximately 40% males and 60% hermaphrodites. To enrich for males, we adopted a mesh filtration approach (Klass and Hirsh 1981). We incubated day 1 adult him-8 worms on a 35-micron nylon mesh suspended over plates containing M9 with NA22 bacteria for 1 hour. Young adult males and larvae are small enough to crawl through the mesh, but not adult hermaphrodites. Worms that crawled through the 35-micron mesh were then placed on a 18-micron nylon mesh for 1 hour. Larvae are small enough to crawl through the 18-micron mesh, but not adult males which were retained on top of the mesh. We scored an aliquot of the resultant adult male enriched preparation to determine the proportion of males in each sample and obtained average proportions of 85% males and 15% hermaphrodites.
Cells were dissociated from male-enriched preparations as previously described (Taylor et al. 2021; Taylor et al. 2024), fixed in methanol, and stored at -20°C for < 1 week. We stained methanol-fixed cells with DAPI and collected diploid cells (2N) using FACS; haploid (1N) germline cells were excluded (Roux et al. 2023). By collecting all diploid cells, rather than targeting populations of neurons marked with fluorescent markers as in our previous profile of L4 larval neurons (Taylor et al. 2021), we were able to capture the entire nervous system with a smaller number of samples. We generated libraries for sequencing using the 10X Genomics platform.
Cell type annotation
Sex-shared neuron clusters were annotated by comparing top markers to the L4 dataset. Male-specific neuron clusters were annotated by expression of fluorescent reporters (Wang et al. 2024)(unpublished data). Annotation of male-specific neurons is ongoing; presumptive male-specific neuron clusters that have not been assigned a cell type ID are labeled as “male.X”.
Thresholding
We performed dynamic thresholding as previously described (Taylor et al. 2021) and selected 4 thresholds of varying stringency.
| Threshold | True positive rate | False discovery rate |
| 1 | 0.806 | 0.227 |
| 2 | 0.755 | 0.190 |
| 3 | 0.635 | 0.155 |
| 4 | 0.499 | 0.129 |
REFERENCES
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Roux AE, Yuan H, Podshivalova K, Hendrickson D, Kerr R, Kenyon C, Kelley D. 2023. Individual cell types in C . elegans age differently and activate distinct cell-protective responses. CellReports. 42(8):112902. doi:10.1016/j.celrep.2023.112902.
Taylor SR, McWhirter RD, Matlock BK, Flaherty DK, Miller DM. 2024. Protocol for isolating specific C. elegans neuron types for bulk and single-cell RNA sequencing. STAR Protoc. 5(4):103439. doi:10.1016/j.xpro.2024.103439.
Taylor SR, Santpere G, Weinreb A, Barrett A, Reilly MB, Xu C, Varol E, Oikonomou P, Glenwinkel L, McWhirter R, et al. 2021. Molecular topography of an entire nervous system. Cell. 184(16):4329–4347. doi:10.1016/j.cell.2021.06.023.
Wang C, Vidal B, Sural S, Loer C, Aguilar GR, Merritt DM, Toker IA, Vogt MC, Cros C, Hobert O. 2024. A neurotransmitter atlas of C. elegans males and hermaphrodites. doi:10.7554/eLife.95402.2. [accessed 2025 Jun 20]. https://elifesciences.org/reviewed-preprints/95402v2.