Dissociation: Single nuclei suspensions were obtained as described in Kalestky R et al., 2018 for cells, with some modifications to further dissociate the cell membranes. After removing most of the M9 solution, worms were quickly washed once in 500ul lysis buffer (200mM DTT, 0.25% SDS, 20mM Hepes pH 8.0, 3% sucrose), and then lysed with 750ul lysis buffer for 5 min for hermaphrodites and 3 minutes for males. Lysis was shorter for males because longer lysis time caused the males to rupture during the downstream M9 washing steps. Cuticle disruption was monitored by examining 1μL of sample under a microscope. When lysis was complete, worms had a slightly blunt head, but were not ruptured. Reaction was stopped by adding 500ul M9 and then did 6 quick washed in M9. Worms were then dissociated by adding 500 ul freshly made 20mg/ml Pronase (resuspended in water) to the worm pellet for 15-20 minutes depending on the lot of Pronase. Males were dissociated in 15 mg/ml Pronase. During the Pronase incubation, worms were dissociated by pipetting vigorously with a P1000 pipette set to 400ul every 45 seconds, rotating between samples for the first 10 minutes, followed by trituration with a P200 pipette set to 200 ul for remaining 5-10 minutes. Worms were quickly spun down between triturations so that the worm pellet size could be monitored. Between pipetting, 2ul of worms were examined on a dissecting scope to assess level of dissociation. Dissociation was continued until large fragments were gone and the pellet of worms was less than 20 ul when quickly centrifuged. We noticed that during this over digestion step, the cell membranes would also digest. To stop the digestion, 750μL of ice-cold 1xPBS/10%FBS was added to the tube. The dissociated nuclei were transferred to a 1ml syringe using a 27-gauge needle. The needle was replaced by a 5uM syringe filter that was pre-wetted with ice cold PBS. The cells were gently passed through the syringe filter directly into the FACS collection tube on ice. Protector RNAse Inhibitor was added to the following solutions: Pronase, PBS-FBS, and PBS-L15-FBS to get final concentration of 2U/ul inhibitor.   Cell sorting and preparation for RNA sequencing: The nuclei were sorted with a Sony SH800S cell sorter fitted with the 100µm nozzle running at a speed of 10-12k events per second using the PE filter set. Nuclei were sorted into 1:1 1xPBS:L15+30%FBS 2U/µl Protector RNAse inhibitor, transferred to a low bind 2 ml tube and centrifuged 9600xg for 5 min at 4C. Once an RFP+ pellet was confirmed under a fluorescent dissection scope, the nuclei were gently resuspended in 1xPBS10%FBS buffer aiming for a final concentration of 1000 nuclei/uL based on the number of nuclei sorted. We targeted 10,000 cells per sample and used the 10X Chromium platform using 10X NextGem v3.1 chemistry. cDNA was amplified for 12 cycles and the index PCR was cycled 12 times. Final libraries were brought to 10 nM concentration. Next-generation sequencing libraries were constructed following the User Guide, which were sequenced on an Illumina NovaSeq 2500 system.