scRNA-seq of Schisotosoma joponicum:18dpi Female
Twenty female C57 mice were infected percutaneously through the abdomen with 80200 cercariae. At each time point (14, 18, 22, and 26 dpi), four mice were sacrificed for the recovery of schistosomes. Worms were collected from mice by perfusing the hepatic portal vein with 4 PBS (Sangon Biotech) and then rinsed in twice in DMEM (5% FBS). Freshly perfused male and female worms, except 14 dpi worms, which are difficult to distinguish gender under a light microscope, were separated by incubation in a 0.25% solution of tricaine for approximately 5 min. FACS sorting was performed as previously described with minor modifications. Worms were washed twice in 8 mL PBS, then triturated for approximately 15 min in 4 mL 0.125% Trypsin-EDTA (in PBS) until the solution became turbid and no large pieces of worms were left. The trypsin was inactivated by adding an 8 mL 10% FBS DMEM. The dissociated worms were then centrifuged at 500 g for 10 min at 4C. Next, the worm cells were resuspended in 1 mL of PBS with 10 L of RQ1 DNAse (Promega M6101) and incubated for 10 min at RT. The worm cells were centrifuged again at 500 g for 10 minutes at 4C. Then the worm cells were resuspended in 1mL of PBS containing Fixable Viability Dye eFluorTM 506 (1L/mL) (eBioscience) and incubated for 30 min at 4 in the dark. The worm cell suspension was centrifuged at 500 g for 10 minutes at 4C, then washed twice in PBS, then filtered through a 40 m cell strainer into a 1275 mm FACS tube. Filtered cells were then sorted on a MoFlo XDP High-Speed Cell Sorter (BECKMAN COULTER) with 405/460/488/532/561/640nm lasers. Sorts were performed with a 100 m nozzle, and cells were sorted into sorting media (0.04% BSA in PBS, pH = 7.40).FACS-sorted cells were centrifuged at 500 g for 10 minutes at 4C and then resuspended in 0.04% BSA in PBS. Libraries were created using a Chromium Controller (10 Genomics) according to manufacturer guidelines and sequenced using a NovaSeq6000 (Illumina) to generate 150-bp paired-end reads.