Illumina NovaSeq 6000 sequencing; Spatially resolved multiomics of human cardiac niches - Visium
Spatially resolved multiomics of human cardiac niches - Visium
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 spatial transcriptomics, ventricle regions, whose thickness were larger than one side of the Visium frame (6 mm), were separated into epicardial and endocardial portions. Samples used for single nuclei isolation and spatial transcriptomics were flash-frozen or frozen in OCT and stored at −80 °C, or formalin-fixed and subsequently embedded in paraffin blocks. All tissues were stored and transported on ice at all times until freezing or tissue dissociation to minimise any transcriptional degradation. Formalin-fixed paraffin-embedded (FFPE) samples: Fresh samples were fixed in >5 times their volume of 4% v/v formalin at ambient temperature for 24 hours before processing to paraffin on a  Tissue-Tek Vacuum Infiltration Processor 5 (Sakura Finetek). FFPE blocks were sectioned at 5 μm using a microtome (Leica RM2125RT). Samples of the microanatomical regions of interest were selected on the basis of morphology with expert review (SYH), orientation (based on HE) and DV200 was obtained using Agilent 2100 Bioanalyzer. In addition, FFPE tissues were checked for possible detachment issues using the 10x Genomics Adhesion test slides. FFPE Visium Spatial Gene Expression (v1 chemistry) was performed following the manufacturer's protocol (10x genomics). H&E stained Visium Gene Expression slides were imaged at 40× on Hamamatsu NanoZoomer S60. cDNA library preparation was were carried out according to the manufacturer's protocol.