Sorting GFP-labeled BAG Neurons

Synchronized young adult hermaphrodites with GFP-labeled BAG neurons (expressing a flp-17p::gfp transgene) were acutely dissociated as described (Kaletsky et al., 2016 (link)). Synchronized adult worms were washed with M9 buffer to remove excess bacteria. The pellet (∼250 μl) was washed with 500 μl lysis buffer (200 mM DTT, 0.25% SDS, 20 mM HEPES pH 8.0, 3% sucrose) and resuspended in 750 μl lysis buffer. Worms were incubated in lysis buffer for 6.5 min at room temperature. The pellet was washed 5 times with M9 and resuspended in 20 mg/ml pronase from Streptomyces griseus (Roche). Worms were pipetted up and down for 12 min at room temperature; then ice-cold PBS buffer containing 2% fetal bovine serum (GIBCO) was added. Cell suspensions were passed over a 5 μm syringe filter (Millipore). The filtered cells were diluted in PBS and sorted using a Sony Biotechnology Synergy High Speed Cell Sorter. Gates for detection were set by comparison to npr-1 cell suspensions prepared on the same day alongside the experimental samples. Positive fluorescent events were sorted directly into Eppendorf tubes containing 10 μL of 0.2% (vol/vol) Triton X-100 and 2 U μl^-1 RNase inhibitor. Six biological replicates were prepared for each genotype, i.e., npr-1(ad609) and arcp-1(db1082); npr-1(ad609) animals. For each replicate sample, approximately 4,000 GFP positive events were collected.