GSM6754000: C57_wt_sham_-491_L5.D703; Mus musculus; RNA-Seq
Cardiomyocytes were isolated from sham and ACF mice from each of the different strains. The hearts were perfused with perfusion buffer (120 mmol/L NaCl, 15 mmol/L KCl, 0.5 mmol/L KH2PO4, 5 mmol/L NaHCO3, 10 mmol/L HEPES, and 5 mmol/L glucose, at pH 7.0) for 8 min and digested with perfusion buffer containing 2% collagenase II for 25 min at 37°C. The right ventricle and atria were removed before the perfused-heart was minced. The cell suspension was then mixed with stop buffer containing 10% fetal bovine serum (FBS). After filtered, the flow-through was spun at 300 rpm for 3 min to pelletize the cardiomyocytes. After calcium re-introduction, only cardiomyocytes with purity and viability (rod-shaped) >95% or 80%, respectively, were used.Cardiomyocytes were isolated from sham and ACF surgery mice for RNA sequence analysis and stored in RNAlater RNA Stabilization Reagent (Qiagen) and flash-frozen in liquid nitrogen for storage. For these studies, the effect of different mtDNA and nuclear background combinations on gene expression was assessed by RNA sequence analysis from RNA isolated with the RNEasy kit (Qiagen). Reverse transcribed cDNA libraries were sequenced with an Illumina HiSeq 2000. Read mapping was conducted with a proprietary algorithm by Expression Analysis (www.q2labsolutions.com), and read counts were used as input for differential expression analysis in DESeq2 version 1.10.1, using default settings. Genes were interpreted as differential expressed if the adjusted p value was reported as less than 0.05. RNA samples were converted into cDNA libraries using the Illumina TruSeq Stranded mRNA sample preparation kit (Illumina # RS-122-2103).