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. Fresh-frozen (FF) samples: Fresh samples were frozen and embedded in optimal cutting temperature medium (OCT) using a dry ice-cooled bath of isopentane at -45°C. OCT-embedded samples were sectioned using a cryostat (Leica CX3050S) and were cut at 10 μm. Samples of the microanatomical regions of interest were selected on the basis of morphology with expert review (SYH), orientation (based on HE) and RNA integrity number was obtained using Agilent 2100 Bioanalyzer. The Tissue Optimization protocol from 10x Genomics was performed to obtain a permeabilisation time of 45 min, and the FF Visium Spatial Gene Expression experiment (v1 chemistry) was performed as per 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.