Single cell immunoglobulin light chain repertoire of Rainbow trout B cells - Fish2
To isolate peripheral blood leukocytes (PBLs) blood was drawn with a heparinized needle from the caudal vein and diluted 10 times with of Leibovitz medium. The resulting cell suspension was placed onto 51% Percoll density cushions collecting the interface cells after centrifugation. Leukocytes were incubated with anti-trout MHC-II APC and YO-PRO in FACS staining buffer. MHC-II+ leukocytes from lymphoid gate were isolated by sorting. A total of 2,500 cells (700 cells/L) from each fish were loaded to chips from the Chromium Single Cell 5? Gel Beads Kit (10x Genomics, CA, USA) and subjected to the Chromium Controller Instrument (10x Genomics) to generate single-cell gel beads in emulsion following manufacturer's instructions. Libraries for sequencing were constructed using specific primers for each rainbow trout IgL subisotype. Libraries were sequenced using a MiSeq instrument (Illumina) with 300PE chemistry.