GSM9409784: lin-39p::RFP unc-3 MNs rep 1; Caenorhabditis elegans; RNA-Seq
KRA547 worms were collected and washed twice. 250 uL pellets of worms were incubated in 500 uL of SDS-DTT buffer (20 mM HEPES, 0.25% SDS, 200 mM DTT, 3% sucrose, pH 8.0) for 6 minutes, then washed 5 times in egg buffer (25 mM HEPES pH 7.3, 118 mM NaCl, 48 mM KCl, 2mM CaCl2, 2mM MgCl2). Worms were then incubated in 500 uL of cold active protease (10 mg/mL in egg buffer, Protease from Bacillus licheniformis, Sigma-Aldrich P4860, with DNAse I 35 U/mL) for 35 minutes at 4 C. During the protease incubations, solutions were triturated by pipetting through a P1000 pipette tip for four sets of 80 repetitions Digestions were quenched by addition of 750 uL of L-15 media supplemented with 10% FBS. Cells and worm debris were pelleted by centrifugation (530 rcf for 5 minutes at 4 C), and then resuspended in 1 mL L-15-10. Cells were separated from worm debris by centrifugation for 2 minutes at 100 rcf at 4 C. The supernatant was collected and filtered through the 35-micron strainer cap of a 5 mL FACS tube (Falcon 352235) The pellet was resuspended a second time in 1 mL of L-15-10, spun at 100 rcf for 2 min at 4 C, and the supernatant was collected and filtered into the same FACS tube to combine with the first extraction. Fluorescently labeled motor neurons were isolated using FACS on a BD FACSAria III equipped with a 70-micron nozzle. GFP+ cells were collected into L-15 media with 33% FBS. Young adult worms remaining on the mesh were collected into M9 and centrifuged at 150 relative centrifugal force (rcf) for 2.5 minutes. Worms were transferred to a 1.6 mL centrifuge tube and centrifuged at 16,000 rcf for 1 minute to form 250 uL pellets. Worm pellets were then treated with 500 µL of SDS-DTT solution (20 mM HEPES, 0.25% SDS, 200 mM DTT, 3% sucrose, pH 8.0) for 6 minutes. Following SDS-DTT treatment, worms were washed five times (each wash: dilute with 1 mL egg buffer, centrifuge at 16,000 rcf for 30 seconds). Worms were then digested in cold-active protease (10 mg/mL, Protease from Bacillus licheniformis, Sigma-Aldrich P4860, with DNAse I 35 U/mL) in egg buffer for 35 minutes at 4°C. Cells were kept at 4°C from protease incubation through all subsequent procedures. During the protease incubations, solutions were triturated by pipetting through a P1000 pipette tip for four sets of 80 repetitions. Dissociation efficiency was monitored at 5-minute intervals with a fluorescence dissecting scope. Digestions were stopped by adding 750 µL L-15 media supplemented with 10% fetal bovine serum (L-15-10). Cells were then centrifuged at 530 rcf for 5 minutes at 4°C. The pellet was resuspended in 1 mL L-15-10, and individual cells were separated from whole worms and debris by centrifuging at 100 rcf for 2 minutes at 4°C. The supernatant was filtered through a 35-micron strainer cap into a 5 mL collection tube (Falcon 352235). The pellet was resuspended a second time, centrifuged (100 rcf for 2 minutes at 4°C), and the resulting supernatant was added to the collection tube. For higher sorting efficiency, samples were diluted with an additional 1 mL of L-15-10. Single-cell suspensions of both genotypes were sorted simultaneously on two BD FACSAria™ III cell sorters faceted with 70-micron diameter nozzles. DAPI was added to the samples (final concentration of 1 µg/mL) to label dead /dying cells. Cells were sorted for three hours under the “4-way Purity” mask into 1.5 mL microcentrifuge tubes containing 200 uL L-15-33 (L-15 medium with 33% fetal bovine serum) and subsequently centrifuged (1200 rcf for 12 minutes at 4°C). Libraries were prepared using P/N 1000075, 1000073, and 120262 following the manufacturer's protocol. Illumina NovaSeq 6000 with 150 bp paired end reads was used for library sequencing