GSM6943641: control rep2; 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  gone through a 70 μm-strainer, and loaded on a Beckman Coulter MoFlo XDP system to sort for eGFP+ or DsRed+ cells. Either 6,000 eGFP+ or DsRed+ cells were sorted into lysis buffer (0.5% Triton X-100, 1 U/μl RNase inhibitor, 2.5 μM Oligo dT primer, 2.5 mM dNTP) on a Beckman Coulter MoFlo XDP system. After vortex and incubation for 3 min at 72℃, total mRNA was reverse-transcribed to cDNA using SuperScript II Reverse Transcriptase (Invitrogen, 18064014). cDNA was then amplified using KAPA HiFi HotStart ReadyMix (Huaruikang, kk2602) and purified using VAHTS DNA Clean Beads (Vazyme, N411). 1 ng cDNA per sample was used to generate library using TruePrep DNA Library Prep Kit V2 for Illumina (Vazyme, TD503) according to the manufacturer's instructions. In brief, 1 ng cDNA was fragmented using TruePrep Tagment Enzyme and then ligated with specific indexes. After amplification using TruePrep Amplify Enzyme, the cDNA library was purified using VAHTS DNA Clean Beads.