GSM4551655: Myo_BCAT_3; Mus musculus; RNA-Seq
Adult CD-1 (ICR) mice were anaesthetized with an i.p injection of ketamine (100 mg/kg) and Xylazil (12.5 mg/kg) 3 days post-surgery. Hearts were quickly dissected and washed in perfusion buffer (120.4 mmol/l NaCl, 14.7 mmol/l KCl, 0.6 mmol/l KH2PO4, 0.6 mmol/l Na2HPO4, 1.2 mmol/l MgSO4•7H2O, 4.6 mmol/l NaHCO3, 10 mmol/l Na-HEPES, 30 mmol/l taurine, 5.5 mmol/l glucose and 10 mM 2,3-Butanedione 2-monoxime). The aorta was then cannulated with a 21-gauge cannula, secured with 3-0 silk suture and perfused with 37◦C, oxygenated perfusion buffer using a Langendorff apparatus (4ml/min). Once all blood was washed from the heart, digestion buffer (200 µg/ml Liberase DH (Roche) in perfusion buffer) was passed through the heart for ~8 minutes (until the hearts appeared waxy in color and flaccid to the touch). During perfusion, atria and excess tissue was removed. After enzymatic digestion, hearts were minced with fine scissors into small pieces and triturated to release cells. Cell isolates were passed through a 100 µm cell strainer and centrifuged at 30xg for 3 minutes at room temperature. Supernatant (containing the non-myocyte cells) was collected and the myocyte pellet was washed with perfusion buffer and re-centrifuged. The supernatant was again collected and the purified myocyte pellet was lysed with 1 ml of Trizol (ThermoFisher). The non-myocyte supernatant was centrifuged at 500xg for 5 minutes and FACS was performed. For enzymatically isolated cells, ribosomal RNA was depleted with Ribo Zero Gold (Illumina), RNA quality ascertained using a MultiNA bioanalyser (Shimadzu) and cDNA generated with SuperScript II Reverse Transcriptase (ThermoFisher). Libraries were created with TruSeq Stranded Total RNA kits (Illumina) and read with HiSeq SR Cluster v4 kit (Illumina) on a HiSeq 2500 sequencer. Each sample contained ~45 million 50-bp single-end reads.
GEO Accession
GSM4551655