Subject Area	Biochemistry, Genetics and Molecular Biology
More specific subject area	C. elegans developmental biology
Protocol name	Cell isolation of C. elegans L1 larvae
Reagents/tools	■90 mm Nematode Growth Media (NGM) agar plates ■NA22 bacteria ■M9 buffer ■5 M NaOH ■Fresh sodium hypochlorite (household bleach) ■SDS-DTT solution (*) ■20 mg/mL Pronase E solution (*) ■Egg buffer (*) ■L-15/FBS medium (*) ■50 mL conical centrifuge tubes ■Glass funnel ■20 µm nylon filter (Sigma, NY2004700) ■32 mm syringe filter (5 μm pore size) (VWR International, 514–4129) ■21G1½ syringe needles (or bigger) (BD Eclipse Needle 21G1½, 305,895) ■1 mL syringe (BD Plastipak, 303,172) ■1.5 mL microcentrifuge tubes ■2 mL microcentrifuge tubes ■Centrifuge (compatible with 50 mL conical centrifuge tubes) ■Benchtop microcentrifuge ■Tube rotator ■Pellet pestle motor (Sigma, Z359971) ■Pellet pestles (Sigma, Z359947-100EA)
	(*) See Appendix A: Supplementary material for a full description of the solution preparation.
Experimental design	This protocol consists of four main steps:
	1.C. elegans culture preparation and growth 2.Synchronization of larvae at L1 arrest 3.Obtaining synchronized populations of specific developmental stages 4.Larvae dissociation and preparation of single-cell suspension
Trial registration
Ethics
Value of the Protocol	■Generation of single cell suspensions can be achieved in a 2.5 h compared to the 3 h of previous protocols, reducing transcriptional effects due to stress and loss of viable cells when FACS sorting. ■Efficient isolation for rare cell types. ■The quality of single cell suspensions allows its use for single cell RNA sequencing.