GSM8173849: medaka_3dpi_rep2; Oryzias latipes; RNA-Seq
To prepare cells for scRNA-seq, adult fish were euthanized by immersion in ice-cold water. After dissection and removal of hearts, the remaining atrium and bulbus arteriosus tissue were cut away with scissors to isolate the ventricle. Ventricles were placed in PBS solution and gently squeezed with forceps to remove residual blood prior to digestion. Whole ventricles were placed Biology Open • Accepted manuscript in 200 µl dissociation solution containing 1mg/mL liberase DH (Millipore Sigma cat# 5401089001) in 1X HBSS. Ventricles were digested at 37°C for 30 min on a benchtop shaker at 250RPM with additional pipetting every 5 min to break apart the tissue. After complete digestion, the reaction was quenched with ice-cold PBS/50% Fetal Bovine Serum and passed through a 40 µM strainer. The cells were then centrifuged at 250 RCF for 10 minutes at 4°C and resuspended in ice-cold PBS with 0.04% Bovine Serum Albumin. Cells were then assessed for viability and dissociation quality using acridine orange and propidium iodide staining with a Nexcelom Cellometer automated cell imager. Each sample viability was assessed to be >85%. Cells were loaded onto a 10X Genomics Chromium controller and processed according to the manufacturer's specifications. cDNA libraries were prepared for sequencing on the Illumina Novaseq platform at a depth of approximately 400M reads/sample. Libraries wer constructed according to the manufacturer's protocol (10X chromium controller)