title: Illumina NovaSeq 6000 paired end sequencing; Mapping the cellular and molecular landscape of cardiac non-myocytes in murine type-2 diabetes
design: Mapping the cellular and molecular landscape of cardiac non-myocytes in murine type-2 diabetes
library_construction_protocol: For single-cell RNA sequencing (scRNAseq) cardiac single cell suspensions were prepared following a modified Langendorff approach described in Farrugia et al., 2021. Briefly, after euthanasia, the thoracic cavity was opened and the inferior vena cava was ligated, before injecting 7mL of cold 'EDTA buffer' (Ackers-Johnson et al., 2016; Farrugia et al., 2021) into the right ventricle for perfusion. Following perfusion, hearts were gently lifted and clamped at the aorta using a haemostat. The hearts were subsequently cut away from the thoracic cavity and mounted onto a 3D-printed platform, such that the apex of each heart was facing up-ward (Farrugia et al., 2021; McLellan et al., 2020). Hearts were then perfused with EDTA buffer at 6mL/min for ~5 minutes. Once hearts had sufficiently cleared with any residual blood, the perfusion rate was reduced to 4mL/min and the perfusate was changed to a collagenase and protease digestion buffer (Ackers-Johnson et al., 2016), for ~15 minutes. All buffers were maintained at 37°C throughout the perfusion process.  Hearts were then further dissociated by gently separating the tissue with forceps before incubating the cell suspension for a further 10-minutes, at 37°C. Cardiac non-myocytes were separated from cardiomyocytes and other cellular debris and subject to antibody/viability-dye staining. Stained non-myocytes consequently underwent fluorescence-activated cell sorting (FACS, BD FACS Aria™ Fusion Flow Cytometer) as described previously (McLellan et al., 2020; Skelly et al., 2018). Live (SYTOX™ Green-), metabolically active (Vybrant™ DyeCycle™ Ruby+) cardiac non-myocyte cells were sorted into endothelial cells (CD31+), leukocytes (CD45+), and resident mesenchymal cells (CD31-CD45-).  Major non-myocyte cell populations were manually counted using a haemocytometer, after which samples were pooled based on their genotype (db/h or db/db). Each pooled sample consisted of ~12,000 cells (ECs; ~10%, RMCs; ~90%) when loaded into the 10X Chromium controller for single cell library preparation (Chromium Single Cell 3' v2, lane 1: pooled db/db non-myocytes [n=4], lane 2: pooled db/h non-myocytes [n=3]). Individual cells were captured and lysed within the 10X controller, after which cDNA was synthesised and amplified for a total of 12 cycles