FUGI_R_D7119553
Schistosoma mansoni
Protocols: Two days after transformation the schistosomula cultured in modified Basch's media at 37°C and 5% CO2 were collected and processed in two separate batches (batch1 and batch2). Schistosomula collected from two different snail batches were considered biological replicates. Data collected as batch3 are 'technical' replicates of batch2 given they were collected on the same day and from the same pool of parasites. In each experiment, approximately 5,000 larvae were pooled in 15ml tubes and digested for 30 min in an Innova 4,430 incubator with agitation at 300 rpm at 37°C, using a digestion solution of 750μg/ml Liberase DL (Roche 05466202001) in PBS supplemented with 20% FBS. The resulting suspension was passed through 70μm and 40μm cells strainers (Falcon). Dissociated cells were spun at 300 rpm for 5 mins and resuspended in 1X cold PBS supplemented with 20% heat inactivated fetal bovine serum (twice). The resulting cell suspension was co-stained with 0.5μg/ml of Fluorescein Diacetate (FDA; Sigma F7378) to label live cells, and 1μg/ml of Propidium Iodide (PI; Sigma P4864) to label dead/dying cells, and sorted into eppendorf tubes using the BD Influx™ cell sorter by enriching for FDA+/ PI- cells. The 10X Genomics protocol (“Single Cell 3' Reagent Kits v2 User Guide” available from https://support.10xgenomics.com/single-cell-gene-expression/index/doc/user-guide-chromium-single-cell-3-reagent-kits-user-guide-v2-chemistry) was followed to create gel in emulsion beads (GEMs) containing single cells, hydrogel beads and reagents for reverse transcription, perform barcoded cDNA synthesis, and produce sequencing libraries from pooled cDNAs. The 10X Genomics protocol (“Single Cell 3' Reagent Kits v2 User Guide” available from https://support.10xgenomics.com/single-cell-gene-expression/index/doc/user-guide-chromium-single-cell-3-reagent-kits-user-guide-v2-chemistry) was followed to create gel in emulsion beads (GEMs) containing single cells, hydrogel beads and reagents for reverse transcription, perform barcoded cDNA synthesis, and produce sequencing libraries from pooled cDNAs.