description: Protocols: For the five aortic stenosis 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. For patients with aortic stenosis the following protocol was used: 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.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2021-12-17
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2022-06-28
attributes.tag: External Id
attributes.value: SAMEA11937491
attributes.tag: INSDC center alias
attributes.value: Institute of Cardiovascular Regeneration Centre of Molecular Medicine Goethe-University Frankfurt
attributes.tag: INSDC center name
attributes.value: Institute of Cardiovascular Regeneration Centre of Molecular Medicine Goethe-University Frankfurt
attributes.tag: INSDC first public
attributes.value: 2021-12-17T12:28:06Z
attributes.tag: INSDC last update
attributes.value: 2022-06-28T14:10:21Z
attributes.tag: INSDC status
attributes.value: suppressed
attributes.tag: Submitter Id
attributes.value: E-MTAB-11268:3013
attributes.tag: age
attributes.value: 67
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell_type
attributes.value: cardiac muscle cell
attributes.tag: common name
attributes.value: human
attributes.tag: developmental stage
attributes.value: adult
attributes.tag: disease
attributes.value: aortic stenosis
attributes.tag: individual
attributes.value: 3013
attributes.tag: isolate
attributes.value: not applicable
attributes.tag: organism part
attributes.value: heart
attributes.tag: sample name
attributes.value: E-MTAB-11268:3013
attributes.tag: sex
attributes.value: male