RNA-seq of Homo sapiens TBX5-FKBP12-F36V: D20 of atrial cardiomyocyte (CM) differentiation with 500 nM dTAGv-1 ligand treated for 24h rep1
Atrial (StemCell Technologies #100-0215) kits were used to differentate iPSCs into cardiomyocytes. DMSO or dTAGv-1 samples were treated at d20 with DMSO or 500 nM dTAGv-1 (Tocris, #6914) ligand for 24 h before harvesting. Cells were dissociated in 0.25% Trypsin-EDTA for 10 mins, followed by quenching in 15%FBS in DPBS. Cells were lifted off the dish and resuspended to single cells by repeatedly using P1000 pipette and then transferred to a 1.5 ml tube and centrifuged at 300 x g for 5 min at room temperature. Pellets were snap frozen in dry ice. Total RNA was isolated using Qiagen RNeasy micro kit (Qiagen #74004) with QIAshredder (Qiagen #79656) and eluted in 25 l water. RNA concentration was determined using Nanodrop and quality was checked using Agilent Bioanalyzer. Messenger RNA was purified from total RNA using poly-T oligo-attachedmagnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers followed by the secondstrand cDNA synthesis. The library was ready after end repair, A-tailing, adapter ligation, size selection, amplification, and purification. The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection.