title: Single-Cell-RNA-Seq of Rainbow trout Head kidney
design: full head kidney tissue from each individual was aseptically extracted and placed into separate falcon tubes filled with 20ml of extraction media (L15[Gibco], 2% FBS and 0.02% EDTA) and kept on ice. Head kidney tissue was gently mashed through a 100 m cell strainer to dissociate cells from tissue with the use of 20 ml of fresh extraction media to pipette cells and tissue through the filter. To isolate leucocytes, the total cell suspension (20 ml) was layered onto a 51% percoll gradient (10 ml) and centrifuged for 30 mins at 400g and 4 C with no brake on. Cells at the interface (mainly leukocytes) were carefully pipetted into a fresh 50 ml tube and washed twice with extraction media, re-pelleting at 400 g and 4 C for 10 mins. Cell viability was assessed using Trypan blue staining and cell counts were done using a Malassez counting chamber. Cells were then diluted to around 1000 cells / L and kept on ice. The scRNA-seq libraries were then prepared using the Single Cell 3 Reagent Kit v3.1 from 10 Genomics (Pleasanton, CA) following the manufacturers protocol. Sequencing of scRNA-seq libraries was done using an Illumina NextSeq500 (San Diego, CA, USA) generating 150 base pair (bp) paired-end reads.