Single nuclei sequencing of human HFpEF (Heart Failure with reduced ejection fraction) and Mice TAC (Transaortic constriction) data
Single nuclei sequencing of human HFpEF (Heart Failure with reduced ejection fraction) and Mice TAC (Transaortic constriction) data
For Mouse hearts, the following protocol was used: cardiac tissues were thawed on ice and cut into small pieces. Minced tissue was pre-digested with a 5-ml enzyme solution of collagenase (2,500 U, Thermo Fisher Scientific) in HBSS+/+ (Gibco) for 10 min at 37 °C in a water bath. After centrifugation at 500g and 4 °C for 5 min, the supernatant was discarded, and nuclei were isolated after cell disruption with a glass dounce homogenizer (five strokes with a loose pestle and ten strokes with a tight pestle)40,41. After filtering (20-µm strainer, pluriSelect), the suspension was centrifuged at 1,000g and 4 °C for 6 min and resuspended in 500 µl staining buffer containing 1% BSA (Sigma-Aldrich), 5 nM MgCl2 (Sigma-Aldrich), 1 mM EDTA (Gibco), 1 mM EGTA (Gibco), 0.2 U µl−1 RNasin Plus Inhibitor (Promega) and 0.1 µg ml−1 Hoechst (Life Technologies) in Dulbecco´s phosphaste buffered saline (DPBS). Hoechst-positive nuclei were separated from cell debris by using the FACSAria Fusion instrument (BD Biosciences) and sorted into staining buffer without Hoechst at 4 °C.   For the two human heart failure samples, biopsies were taken during surgery of the aortic valve, with tissue from the hypertrophied interventricular septum in the left ventricle being collected (75-250 mg per sample; Department of Cardiovascular Surgery, University Hospital Frankfurt am Main, Germany). Informed consent was obtained from all two patients. The study was approved by an institutional review committee of the University Hospital of the Johann Wolfgang Goethe University in compliance with internal standards of the German government and procedures followed were in accordance with institutional guidelines (Application 347/18) and the Declaration of Helsinki. Prior to snRNA-seq, single nuclei were isolated from the hypertrophied interventricular septum in the left ventricle. Cardiac tissues were thawed on ice and cut into small pieces. Minced tissue was then pre-digested in 5 ml enzyme solution of collagenase (2500 U, Thermo Fisher Scientific) in HBSS+/+ (Gibco) for 10 min at 37 °C in a water bath. After centrifugation at 500x g and 4 °C for 5 min, the supernatant was discarded and nuclei were isolated after cell disruption with a glass dounce homogenizer, according to a previously published protocol40. After homogenization of the tissue and filtering through a 20 µm strainer (pluriSelect), the nuclei suspension was centrifuged at 1,000x g at 4°C for 6 min and resuspended in 500 µl staining buffer containing 1 % BSA (Sigma Aldrich), 5 nM MgCl2 (Sigma Aldrich), 1 mM EDTA (Gibco), 1 mM EGTA (Gibco), 0.2 U/µl RNasin®