Danio rerio
Protocols: The guts were dissected and placed in ice cold PBS/5% foetal bovine serum. Single cell suspensions were generated by first passing through a 40 µm strainer using the plunger of a 1 ml syringe as a pestle. These were then passed through a 20 µm strainer before adding 4',6-diamidino-2-phenylindole (DAPI, Beckman Coulter, cat no B30437) to the samples. Three zebrafish, per each condition (i.e. zebrafish intraperitoneally injected with PBS, lyophilised Anisakis simplex or inactivated Vibrio anguillarum), were used to collect the total of 12,000 lck+ cells (4000 per zebrafish) for 10x experiment. Cell were sorted into 1.5 ml Eppendorf tubes containing 20 l 5% FBS in PBS using a BD Influx Index Sorter. Following the sorting, cells were spun down and resuspended in ice cold PBS with 0.04% bovine serum albumin at the concentration of 500 cells/μl. Briefly, cellular suspension was added to the master mix containing nuclease-free water, RT Reagent Mix, RT Primer, Additive A and RT Enzyme Mix. Master mix with cells was transferred to the wells in the row labelled 1 on the Chromium™ Single Cell A Chip (10x Genomics). Single Cell 3' Gel Beads were transferred into the row labelled 2 and Partitioning Oil was transferred into the row labelled 3. The chip was loaded on Chromium™ Controller to generate single-cell GEMs. GEM-RT was performed in a C1000 Touch Thermal cycler (Bio-Rad) at the following conditions: 53°C for 45 min, 85°C for 5 min, held at 4°C. Post GEM-RT cleanup was performed with DynaBeads MyOne Silane Beads (Thermo Fisher Scientific). cDNA was amplified using C1000 Touch Thermal cycler at the following conditions: 98°C for 3 min, 12 cycles of (90°C for 15 s, 67°C for 20 s and 72°C for 1 min), 72°C for 1 min, held 4°C. Amplified cDNA was cleaned with the SPRIselect Reagent Kit (Beckman Coulter) and quality was assessed using 2100 Bioanalyser (Agilent). Libraries were constructed using Chromium™ Controller and Chromium™ Single Cell 3' Library & Gel Bead Kit v2 (10x Genomics) according to the manufacturer's protocol. Briefly, for fragmentation, end-repair and A-tailing cDNA was incubated with Fragmentation Mix in pre-cooled thermocycler at the following conditions: 32°C for 5 min, 65°C for 30 min, held at 4°C. The libraries were then purified using beads. Next adaptor ligation was performed, samples were incubated with Adaptor Ligation Mix at 20°C for 15 min followed by post-ligation clean-up with beads and Sample Index PCR. Again libraries were purified using beads. Following a final Bioanalyzer quality check, the libraries were diluted to the concentration required for sequencing.