characteristics.text: 2025-02-10
characteristics.characteristic: ENA first public
characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: Institute of Cardiovascular Regeneration, Goethe University
characteristics.characteristic: INSDC center name
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS22930880
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-14753:Sample 3
characteristics.characteristic: Submitter Id
characteristics.text: 12
characteristics.unit: week
characteristics.characteristic: age
characteristics.text: ArrayExpress
characteristics.characteristic: broker name
characteristics.text: cardiac muscle cell
characteristics.characteristic: cell_type
characteristics.text: not collected
characteristics.characteristic: collection date
characteristics.text: house mouse
characteristics.characteristic: common name
characteristics.text: Protocols: 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® Plus Inhibitor (Promega) and 0.1 µg/ml Hoechst (Life Technologies) in DDPBS. Then, Hoechst-positive single nuclei were separated from cell debris by FACS using the FACS ARIA Fusion instrument (BD Biosciences) and sorted into staining buffer without Hoechst at 4 °C. Nuclei suspensions were loaded on a 10X Chromium Controller (10X Genomics) according to manufacturer’s protocol based on the 10X Genomics proprietary technology. All snRNA-seq libraries were prepared using Chromium Single Cell 3′ v3 Reagent Kit (10X Genomics) according to manufacturer’s protocol. Briefly, the initial step consisted in performing an emulsion where individual nuclei were isolated into droplets together with gel beads coated with unique primers bearing 10X cell bar-codes, UMI (unique molecular identifiers) and poly(dT) sequences. Reverse transcription reactions were engaged to generate barcoded full-length cDNA followed by the disruption of emulsions using the recovery agent and cDNA clean up with DynaBeads MyOne Silane Beads (Thermo Fisher Scientific). Total cDNA was amplified using a Biometra Thermocycler TProfessional Basic Gradient with 96-Well sample block (98 °C for 3 min; cycled 14×: 98 °C for 15 s, 67 °C for 20 s, and 72 °C for 1 min; 72 °C for 1 min; held at 4 °C). Amplified cDNA product was cleaned with the SPRIselect Reagent Kit (Beckman Coulter). Indexed sequencing libraries were constructed using the reagents from the Chromium Single Cell 3′ v3 Reagent Kit, as follows: fragmentation, end repair and A-tailing; size selection with SPRIselect; adaptor ligation; post-ligation cleanup with SPRIselect; sample index PCR and cleanup with SPRI select beads.
characteristics.characteristic: description
characteristics.text: adult
characteristics.characteristic: developmental stage
characteristics.text: aortic stenosis
characteristics.characteristic: disease
characteristics.text: wild type genotype
characteristics.characteristic: genotype
characteristics.text: not collected
characteristics.characteristic: geographic location (country and/or sea)
characteristics.text: 3
characteristics.characteristic: individual
characteristics.text: not applicable
characteristics.characteristic: isolate
characteristics.text: Mus musculus
characteristics.characteristic: organism
characteristics.text: Mus musculus
characteristics.characteristic: organism
characteristics.text: heart
characteristics.characteristic: organism part
characteristics.text: Mus musculus
characteristics.characteristic: scientific_name
characteristics.text: male
characteristics.characteristic: sex
characteristics.text: C57Bl/6J
characteristics.characteristic: strain
characteristics.text: Sample 3
characteristics.characteristic: title