Single cell transcriptome profiling of the Atlantic cod immune system

Single cell RNA sequencing of peripheral blood leukocytes and spleen cells from healthy Atlantic cod (Gadus morhua).The Atlantic cod from one single breeding family (bred from 1 female and 2 males) were originally supplied as juveniles by NOFIMA (Tromsø, Norway) where there is a national breeding program for cod. The cod were reared at the NIVA Research Facility at Solbergstrand (near Oslo), Norway (FOTS ID 12336).Blood and spleen samples were taken from two specimens of nonvaccinated, 2-year-old Atlantic cod; 1 male (fish 1, 47cm, 0.99kg) and 1 female (fish 2, 52cm, 1.77kg). Blood samples were collected from the vena caudalis withheparinized syringes. The spleens were removed and placed in Leibovitz L-15þ (L-15 (BioWhittaker) (adjusted to 370 mOsm by adding 5% (v/v) of a solution consisting of 0.41 M NaCl, 0.33 M NaHCO3 and 0.66% (w/v) D-glucose)) and transported on ice. Spleen cell suspensions were obtained by gentlyforcing the tissue through a cell strainer (Falcon, 100 µm). Blood samples of 0.7ml were diluted in L15þ to a total volume of 5 ml. The blood cell suspensions were placed on discontinuous Percoll gradients (3 ml 1.070 g/ml overlaid with 2.5 ml 1.050 g/ml) and centrifuged for 40 min at 400xg and4oC. A peripheral blood leukocyte (PBL) fraction was collected from the interface of the two Percoll densities, including the downward density layer, and washed twice by diluting the suspension in L-15þ and centrifuging at 300xg for 7 min at 4oC.Spleen, blood and PBL suspensions were further separated into sub-populations on a FACS Aria II flow cytometer (Flow Cytometry Core facility at Oslo University Hospital) gated on the forward scatter (FSC, cell size) vs side scatter (SSC, granularity) plot. The sorted populations were examined bymicroscopy after cytospin and staining (supplementary Figure 1). Sub-populations of potential interest from the blood (B1, containing a large lymphocyte population), the spleen (S3, containing a large myeloid cell population) and from PBLs (P1, P2 and P3) as well as unsorted spleen and PBL cell suspensions were subjected to scRNA-seq using the Drop-seq protocol (Macosko, Basu et al. 2015, Dolomite, UK for droplet generation) and NextSeq500 platform with a 75 bp kit, high output mode, with paired end reads (20 bp for R1 and 60 bp for R2). R1 custom sequencing primer: (GCCTGTCCGCGGAAGCAGTGGTATCAACGCAGAGTAC. R2 standard illumina primer.