Mus musculus
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 three 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 five 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.
INSDC center name
Institute of Cardiovascular Regeneration Centre of Molecular Medicine Goethe-University Frankfurt
Submitter Id
E-MTAB-13264:Mice-CTRL-2
age
0.25
broker name
ArrayExpress
cell_type
cardiac muscle cell
collection date
not collected
common name
house mouse
developmental stage
adult
disease
normal
geographic location (country and/or sea)
not collected
isolate
not applicable
organism part
heart
sample name
E-MTAB-13264:Mice-CTRL-2
scientific_name
Mus musculus
sex
male