GSM4040775: Sham; Mus musculus; RNA-Seq
Acute myocardial infarction (MI) was modeled in C57BL6/J wild type (WT) 8-10-week-old male mice (The Jackson Laboratories and confirmed via echocardiographic analysis, similar to previously described (Duan, et al., 2017). A left thoracotomy was performed via the fourth intercostal space and the lungs retracted to expose the heart. After opening the pericardium, the left anterior descending coronary artery was ligated with 7-0 silk suture approximately 2 mm below the edge of the left atrial appendage. Ligation was considered successful when the anterior wall of the left ventricle turned pale. The lungs were inflated by increasing positive end-expiratory pressure and the thoracotomy site closed in layers with 6-0 suture. Animals were maintained on a 37 °C heating pad until recovery and for 2 h after surgery. Another group of mice underwent sham ligation, with a similar surgical procedure without tightening the suture around the coronary artery. Mice with an estimated pressure gradient across the aortic constriction below 40 mmHg were not included in the experiments. Hearts were collected at 14 days post-Sham surgery (n=4, pooled) or MI (n=1), perfused with ice-cold PBS to remove red blood cells followed by perfusion with 50 mM KCl to arrest the heart in diastole and then fixed for 4 hours in freshly prepared 4% PFA at 4 °C, rinsed with PBS and cryoprotected in 30% sucrose/PBS overnight before embedding in OCT (Tissue-Tek). DropSeq was performed as previously described (Macosko, et al., 2015). The quantity and quality of cDNA was measured using an Agilent Bioanalyzer hsDNA chip. To generate a library cDNA was fragmented and amplified (12 cycles) using the Nextera XT DNA Sample prep kit with three separate reactions of 600, 1,200 and 1,800 pg input cDNA. The quantity and quality of cDNA was measured using an Agilent Bioanalyzer hsDNA chip. To generate a library cDNA was fragmented and amplified (12 cycles) using the Nextera XT DNA Sample prep kit with three separate reactions of 600, 1,200 and 1,800 pg input cDNA. The libraries were pooled and purified twice using 0.7X volume of SPRIselect beads. The purified libraries were quantified using an hsDNA chip and were sequenced on an Illumina HiSeq 2500 using the sequencing parameters described in the DropSeq protocol. DropSeq was performed as previously described (Macosko et al., 2015).