title: HCAHeartST13177112
organism: Homo sapiens
description: Protocols: Hearts were acquired after circulatory death (DCD) or after brain death (DBD). For DCD donors after confirmation of death there was a mandatory five-minute stand-off prior to sternotomy. In all cases the aorta was cross-clamped and cold cardioplegia solution was administered to the aortic root prior to cardiotomy. AV1, AV3, AV10, AV13 and AV14 were procured in the standard fashion and then immediately preserved with and transported on a hypothermic perfusion machine. AH2 was similarly preserved, but with immediate normothermic perfusion. It then underwent 4 hours of normothermic perfusion before samples were taken. For single nuclei sequencing, all donor samples were full-thickness myocardial tissues from the SAN, AVN, left and right atrium, left and right ventricles, interventricular septum and apex. Single nuclei were obtained from flash-frozen tissues using sectioning and mechanical homogenization. 5-10 mm thickness frozen tissues were first sectioned with cryostat in a 50 μm thickness section. All sections from each sample were homogenised using a 7 ml glass Dounce tissue grinder set (Merck) with 8–10 strokes of a loose pestle (A) and 8–10 strokes of a tight pestle (B) in homogenization buffer (250 mM sucrose, 25 mM KCl, 5 mM MgCl2, 10 mM Tris-HCl, 1 mM dithiothreitol (DTT), 1× protease inhibitor, 0.4 U μl−1 RNaseIn, 0.2 U μl−1 SUPERaseIn, 0.1% Triton X-100 in nuclease-free water). Homogenate was filtered through a 40-μm cell strainer (Corning). After centrifugation (500g, 5 min, 4 °C) the supernatant was removed and the pellet was resuspended in storage buffer (1× PBS, 4% bovine serum albumin (BSA), 0.2U μl−1 Protector RNaseIn). Nuclei were stained with 7-AAD Viability Staining Solution (BioLegend) and positive single nuclei were purified by fluorescent activated cell sorting (FACS) using MA900 Multi-Application Cell Sorter (Sony). Nuclei were further processed for 10x multiome paired RNA and ATACseq using the Chromium Controller (10X Genomics) according to the manufacturer's protocol. ATAC library preparation was were carried out according to the manufacturer's protocol.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2023-06-13
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2023-06-13
attributes.tag: External Id
attributes.value: SAMEA113413090
attributes.tag: INSDC center alias
attributes.value: Wellcome Sanger Institute
attributes.tag: INSDC center name
attributes.value: Wellcome Sanger Institute
attributes.tag: INSDC first public
attributes.value: 2023-06-13T12:22:47Z
attributes.tag: INSDC last update
attributes.value: 2023-06-13T12:22:47Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-12919:HCAHeartST13177112
attributes.tag: age
attributes.value: 40 to 45
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: common name
attributes.value: human
attributes.tag: developmental stage
attributes.value: adult
attributes.tag: disease
attributes.value: normal
attributes.tag: genotype
attributes.value: wild type genotype
attributes.tag: individual
attributes.value: AH2
attributes.tag: organism part
attributes.value: sinoatrial node
attributes.tag: sample name
attributes.value: E-MTAB-12919:HCAHeartST13177112
attributes.tag: scientific_name
attributes.value: Homo sapiens
attributes.tag: sex
attributes.value: male