RNA-seq of cardiac-specific Cdk1 knockout mice at 4 days after experimental myocardial infarction
RNA-seq of cardiac-specific Cdk1 knockout mice at 4 days after experimental myocardial infarction
Adult mice at 4d post-MI were sacrificed by cervical dislocation following ethical guidelines, and the hearts were immediately excised and rinsed in ice-cold PBS to remove any remaining blood. Individual left ventricular (LV) specimens were rapidly cut into 4-6 pieces, snap frozen in liquid nitrogen, and stored at -80°C. All animal usage in this study was in accordance with approved institutional animal care guidelines of the University Health Network (AUP 1379; Canadian Council in Animal Care). All animals used in this study were male and 10-12 weeks old (19-23g) at the beginning of experimentation. All experiments used isogenic littermate controls of matched age and sex. After weaning, experimental male mice were housed in groups of 3-5 animals in mechanically ventilated cages (600 cm2; changed fortnightly) environmentally enriched by bedding and nesting materials and crawl tubes. Animals were held in a temperature-controlled environment at 19-22°C on a diurnal 12 hour light cycle. Mice were provided free access to standard nonmedicated pelleted laboratory rodent chow containing minute levels of phytoestrogens (Tekland irradiated LM-485 mouse/rat Diet 7912; Envigo, Madison WI , USA; ). Tap water was supplied ad libitum from a portable water faucet. To create a MI, permanent ligation of the left descending coronary artery was performed in in 10-12 weeks old male Cdk1KOc and control mice. At the time of surgery and at 24h post MI, a single subcutaneous dose of buprenorphine (30 mg/g body weight) (B9275; Sigma-Aldrich) was given. To prepare total RNA extracts, frozen LV tissue (80-100 mg wet weight) was transferred to a 2.0 ml reaction tube and mixed with 1.0 ml ice-cold Trizol reagent (no. 15596026). Then, heart tissue samples were lysed at 4oC for 3 minutes at a frequency of 30/s using the TissueLyser II (Qiagen). Samples were incubated on ice for 30 minutes, vortexing every 5 minutes for 30 seconds. Samples were then subjected to 10 cycles of 3 seconds on/1 second off sonification at 30% amplitude on ice, and incubated for 10 minutes, vortexing every 5 minutes for 30 seconds. After centrifugation at 12,700 rpm for 30 minutes at 4oC, RNA was isolated and purified from the supernatant according to the manufacturer's instructions (Trizol; Thermo Fisher Scientific). RNA quality was assessed by 260/280 and 260/230 absorption ratios employing a Nanodrop spectrophotometer (NanoDrop; Thermo Fisher Scientific). Library construction and RNA-seq of the RNA samples were performed at the Princess Margaret Genomics Centre, 101 College St., 9-601, Toronto, ON, M5G 1L7, Canada, using an Illumina NextSeq 2000.