title: GSM8727400: Caenorhabditis elegans, YA Males, Neurons, rep 1; Caenorhabditis elegans; RNA-Seq
library_name: GSM8727400
library_construction_protocol: To generate pure hermaphrodites populations C. elegans strain OH10690 with the genotype rab-3p(prom1)::2xNLS::TagRFP] IV was used. To generate male populations, C. elegans strain MOS264 with the genotype rab-3p(prom1)::2xNLS::TagRFP] IV; dpy-28(y1) III; him-8(e1489) IV was employed. This MOS264 strain harbors a temperature-sensitive mutation (y1) in the dosage compensation gene, dpy-28 along with him-8(e1489) mutation and a pan-neuronal RFP. This resulted in male population of ≥98% purity. After obtaining the worms of the sex and Young adult age, they were washed 10 times at 1300 rpm for 2 minutes with M9 buffer to get rid of any adhering bacteria. The worms were then washed with ddH2O. The worm pellets were then subjected to single cell dissociation using protocols described earlier with slight modifications. For every 0.5 ml of worm pellet 1 ml of freshly thawed sterile SDS-DTT solution (200 mM DTT, 0.25% SDS, 20 mM HEPES, 3% sucrose, pH 8.0, thawed freshly) was added and incubated at room temperature for 4 min with gentle mixing every 1 minute. Immediately after SDS-DTT treatment, worms were pelleted at 3,000 rpm for 1 min and washed 5 times with 10 ml egg buffer (118 mM NaCl, 48 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 25 mM HEPES, pH 7.3). 0.5 ml aliquots of worm pellet were distributed to 1.6 mL microcentrifuge tubes. 1 mL freshly prepared 15 mg/mL Pronase-E was added to each tube and flicked to mix and then rotated at 20°C for 5 minutes. Using a glass pasteur pipette, worm suspensions were combined from multiple tubes and were transferred to a 7 mL dounce homogenizer (pestle clearance 0.02−0.056 mm, Kimble Chase). Pronase E was added to a final ratio of 4:1 relative to the worm pellet. If the worm pellet was smaller than 0.5 mL a 2mL glass homogenizer was used. The animals were disintegrated to single cells using type “B” plunger for 4-5 minutes at room temperature and then continued on ice gently. We note that homogenizing on ice increased cell survival. The status of dissociation was monitored under a dissecting microscope at 5-minute intervals. Dissociation was stopped when most of the animals were dissociated but a few whole or fragmented animals were still present. Using a glass pasteur pipette the cell suspension was aliquoted into 1.6 mL microcentrifuge tubes that were centrifuged at 5,000 x g for 5 minutes at 4 °C. Supernatant was removed and the cell pellet was washed 3 times with 1 mL ice-cold egg buffer per wash at 5,000 x g for 5 minutes at 4 °C. The cells pellet was resuspended in 1 ml ice-cold egg buffer and was passed through 5 μm syringe filter gently. Cell suspension was taken on ice to Fluorescence Activated Cell Sorting (FACS) sorting immediately. Single cell suspension from the desired strain were subjected to FACS on a BD FACSAria™ III Cell Sorter using a 70-micron nozzle. eFlour 450 dye (final concentration of 1 μl/mL) was used to discriminate live and dead cells. The cells were gated for side scatter (SSC)-Area vs forward scatter (FSC)-Area, then single cells were selected from doublet using FSC-Height vs FSC-Area. Finally, RFP+ cells were gated and sorted under the “4-way Purity” mask from this subpopulation. Wild-type him-5(e1490) without any RPF+ cells was used as control for gating. Sorted cells were collected in 1.6 mL microcentrifuge tube containing 500 µl L-15-20 (L-15 medium containing 20% fetal bovine serum). The suspension was centrifuged at 5,000 x g for 10 minutes at 4 °C, resuspended in 50-80 µl of L-15-20 and counted using a hemocytometer. Single-cell capture and library preparation were performed following standard 10x Genomics protocols. A target of 10,000 cells per sample was set, and libraries were prepared using the Single Cell 3' v3 (Dual Index) chemistry kit.
identifiers.namespace: GEO
identifiers.id: GSM8727400_r1