GSM5597246: Sham-dantrolene biological replicate 1; Mus musculus; RNA-Seq
Under anesthesia with an anesthesia machine (R580, RWD Life Science, China), the mice were weighed and sacrificed by cervical dislocation. We Started below the xiphoid, made a V-shaped thoracotomy, opened the thorax, and exposed the heart. The heart was arrested in diastole with 1% KCl in 0.01M PBS, quickly removed, rinsed with cold PBS, and blotted dried. Total RNA was treated with RQ1 DNase (Promega) to remove DNA. The quality and quantity of the purified RNA were determined by measuring the absorbance at 260nm/280nm (A260/A280) using smartspec plus (BioRad). RNA integrity was further verified by 1.5% Agarose gel electrophoresis. For each sample, 1µg of total RNA was used for RNA-seq library preparation. Polyadenylated mRNAs were purified and concentrated with oligo(dT)-conjugated magnetic beads (Invitrogen) before being used for directional RNA-seq library preparation. Purified mRNAs were fragmented at 95℃ in fragmentation buffer followed by end repair and 5' adaptor ligation. Then reverse transcription was performed with RT primer harboring 3' adaptor sequence and randomized hexamer. The cDNAs were purified and amplified and PCR products corresponding to 200-500 bps were purified, quantified and stored at -80℃ before sequencing. For high-throughput sequencing, the libraries were prepared following the manufacturer's instructions and applied to Illunima HiSeq X Ten system for 150 nt paired-end sequencing