Femtosecond laser microdissection for isolation of regenerating C. elegans neurons for single-cell RNA sequencing
Our understanding of nerve regeneration can be enhanced by delineating its underlying molecular activities at single-neuron resolution in model organisms such as Caenorhabditis elegans. Existing cell isolation techniques cannot isolate neurons with specific regeneration phenotypes from C. elegans. We present femtosecond laser microdissection (fs-LM), a single-cell isolation method that dissects specific cells directly from living tissue by leveraging the micrometer-scale precision of fs-laser ablation. We show that fs-LM facilitates sensitive and specific gene expression profiling by single-cell RNA sequencing (scRNA-seq), while mitigating the stress-related transcriptional artifacts induced by tissue dissociation. scRNA-seq of fs-LM isolated regenerating neurons revealed transcriptional programs that are correlated with either successful or failed regeneration in wild-type and dlk-1 (0) animals, respectively. This method also allowed studying heterogeneity displayed by the same type of neuron and found gene modules with expression patterns correlated with axon regrowth rate. Our results establish fs-LM as a spatially resolved single-cell isolation method for phenotype-to-genotype mapping.
Zhao, Peisen; Mondal, Sudip; Martin, Chris; DuPlissis, Andrew; Chizari, Shahab; Ma, Ke Yue; Maiya, Rajani; Messing, Robert O.; Jiang, Ning; and Ben-Yakar, Adela, "Femtosecond laser microdissection for isolation of regenerating C. elegans neurons for single-cell RNA sequencing" (2023). School of Graduate Studies Faculty Publications. 62.