title: GSM7082521: N2 no treatment, replicate 2, scRNA-Seq; Caenorhabditis elegans; RNA-Seq
library_construction_protocol: A synchronous population of L1 worms was obtained by double bleaching gravid N2 C. elegans with hypochlorite followed by 4 washes in S-buffer. The released eggs were then allowed to hatch in the absence of food in S-buffer over a period of 18 hours. Approximately 100,000 synchronized L1 worms were then grown in NGM plates seeded with HT115 bacteria at 20℃ for approximately 55h. 55h post-seeding, worms were staged under a microscope to ensure that they have reached the young adult stage. Young adult worms were then harvested in S-buffer and then centrifuged at 1,300g for 1 min. The worm pellet was washed until the suspension was no longer turbid (2-3 times) and then transferred to a 1.5mL Eppendorf tube. The cuticle was then disrupted by incubating the worms in 200μL SDS-DTT (20mM HEPES pH8.0, 0.25% SDS 200 mM DTT, 3% sucrose) for 4 minutes. Immediately after SDS-DTT treatment, 800 mL of egg buffer was added to the treated worms, the worms were centrifuged, the supernatant was aspirated and the worm pellet was washed 5 times in egg buffer (118 mM NaCl, 48 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 25 mM HEPES, at osmolarity of 340 Osm). After the final wash, egg buffer was added to a final volume of 1mL and the worm solution was then transferred to a 15-mL conical tube. 500μL of 350 units/mL Pronase (EMD Millipore Corp) was added and the worms were then dissociated into single cells by passing them through a 21-gage needle about 20 times. The worm/cell lysate was centrifuged at 4℃ for 1 min at 200g and then most of the supernatant, containing dissociated cells, was transferred to a new 15-mL conical tube leaving behind enough liquid for a second round of dissociation. After passing the worm lysate through the needle for a second time, the samples were centrifuged (4℃ for 1 min at 200g) and then the supernatant was transferred to the same tube containing the cells from the first transfer. The cells were then centrifuged at 4℃ for 5 min at 500g and the cell pellet was washed 3 times in egg buffer containing 1% BSA gently pipetting the cells with wide-end tips. Finally, to separate single cells from bigger chunks of tissue, the cell suspension was gently passed through a 10μm filter. For single cell capture, 14,000 C. elegans cells were mixed with the reverse transcriptase solution and then loaded onto each channel of the 10x Chromium Controller. The libraries were then built following the Chromium Next GEM Single Cell Kits v3.1 published protocols and then sequenced on an Illumina NextSeq 500 platform.