GSM7012024: clofv087b/+ sham; Danio rerio; RNA-Seq
For each sample, 15 adult ventricles were collected and placed in ice-cold perfusion buffer (130 mM NaCl, 5 mM KCl, 0.5 mM NaH2PO4, 10 mM HEPES, 10 mM Glucose, 10 mM 2,3-butanedionemonoxime, 10 mM taurine, 5 mM MgCl2, adjusting pH to 7.8 with NaOH). The ventricles were gently torn up, transferred, and digested in the digestion buffer (10 mg/ml protease, 30 μg/ml DNase I in perfusion buffer) for 3 h at 4℃ with constant gentle agitation by using a rotator. The digestion was then neutralized with 10% FBS (fetal bone serum) and the disassociated cells were filtered through a 100 μm-strainer to remove large clumps and pelleted at 500 x g for 10 min at 4℃. The pellet was re-suspended in Hank's balance salt solution (HBSS). The disassociated cells were stained with Hochest 33342 (1:300 diluted in HBSS),  gone through a 70 μm-strainer, and loaded on a Beckman Coulter MoFlo XDP system to remove cell debris and sort for single cells. The sorted single cells were pelleted at 1000 x g for 5 min at 4℃. The pellet was resuspended in HBSS. Cell viability and concentration were measured on a Countstar Rigel system (ALIT Life Science). Samples with more than 85% cell viability were qualified for single-cell loading. Around 12,000 cells per sample were loaded into 10x Genomics Chromium chip. Single-cell RNA-seq libraries were generated using Chromium Next GEM Single Cell 3' Kit v3.1 (10x Genomics, 1000269) according to the manufacturer's instructions.