GSM8284514: male parasites, control RNAi, scRNAseq, rep1; Schistosoma mansoni; RNA-Seq
100 male worms from each group were suspended in a 0.5% solution of Trypsin/EDTA (Sigma T4174) in PBS before rinsing in PBS for triple times. The worms were then triturated for approximately 10 minutes at room temperature until the solution became turbid and no large pieces of worms were left. The trypsin was inactivated by adding an equal volume of Basch media. The dissociated worms were then centrifuged at 500 g for 10 min at 4°C. Next the worms were resuspended in 1 ml of Basch medium followed by filtering through 100uM cell strainer to remove big chunks. The filtered collections were treated with 10 μL of RQ1 DNAse (Promega M6101) and incubated for 10 minutes at RT. After adding 3mL PBS, the dissociated worms were centrifuged again at 500 g for 10 minutes at 4°C. The cells were then resuspended in 1mL of staining media (0.2% BSA, 2mM EDTA in PBS, pH 7.40) and incubated in Hoechst 33342 (18 μg/ml) (Sigma B2261) for 30min at RT in the dark. The worms were centrifuged once again at 500 g for 10 minutes at 4°C. Worms were then resuspended in 1mL of staining media containing Hoechst 33342 (18 μg/ml) and propidium iodide (1 μg/ml) (Sigma-Aldrich P4170) and then filtered through a 100 μm cell strainer into a 12x75mm FACS tube prior to sorting. Filtered cells were then sorted on a FACSAria II custom (BD Biosystems) with 305/405/488/561/633nm lasers. Live single cells (PI negative, singlet by comparing forward scatter height to forward scatter width) were sorted using a 100 μm nozzle and cells were sorted into sorting media (0.2% BSA in PBS, pH 7.4). A Hoechst threshold was applied to exclude debris and improve the efficiency of sorting. single-cell RNAseq libraries were prepared using a 10x genomics Chromium Controller according to the manufacturer's protocol