RT Journal Article SR Electronic T1 Femtosecond laser microdissection isolating regenerating C. elegans neurons for single cell RNA sequencing JF bioRxiv FD Cold Spring Harbor Laboratory SP 2021.01.21.427576 DO 10.1101/2021.01.21.427576 A1 Peisen Zhao A1 Chris Martin A1 Ke-Yue Ma A1 Ning Jiang A1 Adela Ben-Yakar YR 2021 UL http://biorxiv.org/content/early/2021/01/21/2021.01.21.427576.abstract AB Our understanding of nerve regeneration can be enhanced by delineating its underlying molecular activities at single neuron resolution in small model organisms such as Caenorhabditis elegans. Existing cell isolation techniques cannot isolate regenerating neurons from the nematode. We present femtosecond laser microdissection (fs-LM), a new single cell isolation method that dissects intact cells directly from living tissue by leveraging the micron-scale precision of fs-laser ablation. We show that fs-LM facilitated sensitive and specific gene expression profiling by single cell RNA-sequencing, while mitigating the stress related transcriptional artifacts induced by tissue dissociation. Single cell RNA-sequencing of fs-LM isolated regenerating C. elegans neurons revealed transcriptional program leading to successful regeneration in wild-type animals or regeneration failure in animals lacking DLK-1/p38 kinase. The ability of fs-LM to isolate specific neurons based on phenotype of interest allowed us to study the molecular basis of regeneration heterogeneity displayed by neurons of the same type. We identified gene modules whose expression patterns were correlated with axon regrowth rate at a single neuron level. Our results establish fs-LM as a highly specific single cell isolation method ideal for precision and phenotype-driven studies.Competing Interest StatementThe authors have declared no competing interest.