GSM7036441: blood_ctrl; Scophthalmus maximus; RNA-Seq
Five turbots were randomly selected to collect blood for cell suspension preparation. Blood were added into anticoagulant (Solarbio) to inhibit coagulation. Cell suspensions were washed with 0.04% BSA/PBS and submitted to density gradient centrifugation at 400 × g, 30 min. Leukocytes were collected from the lower cell layer in the middle of discontinuous Percoll (1.057 g/mL and 1.077 g/mL) (BioDee) and washed with PBS twice at 600 × g. Single-cell libraries were prepared using the Chromium 3' v3 platform (10X Genomics). Briefly, blood leukocytes were diluted into 700 – 1200 cells/μl and combined with partitioning oil, and gel beads mix to generate gel beads in emulsion (GEM) on a microﬂuidic chip. After RNA reverse transcription and cDNA barcoding, GEMs were lysed and subjected to cDNA cleanup, cDNA ampliﬁcation, cDNA fragmentation, and library adaptor ligation.