Illumina HiSeq 4000 sequencing; Single cell RNA-Seq of the mouse heart in response to chronic stress
Single cell RNA-Seq of the mouse heart in response to chronic stress
Mice were fed standard diet ad libitum prior to isolation of tissues. Treatment of the mice was achieved by subcutaneous implantation of osmotic pumps (Alzet, model 2002) loaded with angiotensin II or vehicle. AngII was administered at 1.5mg/kg/day for two weeks after implantation of the pump. Male and female mice were sacrified at 10 weeks of age.   For simultaneous isolations of cardiomyocytes and non-myocytes, we adapted a protocol previously described for perfusion-based dissociation of the heart (Ackers-Johnson et al., 2016). Recipes for all buffers used can be found in the aforementioned protocol with the following modifications, 2-3, butanedione monoxime (BDM) was replaced with 5mM blebbistatin (SigmaAldrich) and [0.1U/µL] murine RNase inhibitor (NEB) was also supplemented.  One-hour prior to tissue harvest, mice were administered an intraperitoneal heparin injection at 1 mg/kg bodyweight. Following euthanasia of mice by CO2 asphyxiation, thoracic cavities were opened and inferior vena cava cut to allow exsanguination, before injecting 7 ml of EDTA buffer le. Following perfusion, aortas were clamped using a hemostat and cut to extract hearts. The hemostat was subsequently placed on a 3D-printed platform enabling hearts to be suspended above a waste fluid collection reservoir, with apex of the heart pointing upwards. To perfuse fluid, a hypodermic needle, connected to a peristaltic pump through the 3D-printed platform  which enabled it to be orientated above the heart, was inserted to the left ventricular chamber through the cardiac apex. Once the hearts were cannulated, EDTA buffer was pumped through using a peristaltic pump (~1ml/heart/min) until blood cleared (~5 min) before perfusion with enzyme digest cocktail for 15 minutes, during which time cardiac tissue was basted with fresh digestion buffer to prevent desiccation of the tissue. All buffers were maintained at 37°C during perfusion.   Following tissue digestion, cardiac ventricles were isolated by removing aorta and atria, and the ventricular digestion was finished by gently pulling apart the tissue before a 5-minute incubation at 37oC followed by trituration with a 1000 µL pipette. The cell suspension was then passed through a 250 µM filter to remove large undigested debris and brought to 15 mL with cold perfusion buffer supplemented with 5% FBS in a conical tube. Three consecutive minute-long centrifugation sedimentations at 50g, 4C were performed to separate cardiomyocytes and non-myocytes based on cell mass. The supernatant from each sedimentation step was collected and pooled in a 50 mL tube which was then centrifuged at 400g, 4C for 5 minutes to pellet the non-myocyte fraction of cells. Whole intact non-myocyte cells then underwent antibody and viability dye staining before flow cytometry or fluorescence-activated cell sorting (FACS), as described previously.  Cardiomyocyte nuclei were isolated by swelling cardiomyocytes in a hypotonic solution (91 mM NaCl, 5.3 mM KCl, 0.5 mM MgCl2, 0.293 mM CaCl2, 10 mM glucose, 10 mM HEPES at pH7.4) before triturating cells using a 30-gauge hypodermic needle to mechanically fragment cells. Nuclei were then isolated by filtration through a 20-µm cell strainer (pluriStrainer) to further fragment cells and exclude large debris. Finally, cell fragments were centrifuged at 1000g for 5 minutes before staining with DAPI, followed by FACS for isolation of nuclei.   Intact non-myocyte cells and cardiomyocyte nuclei from each heart were mixed and used for single cell transcriptomic library preparation on the Chromium controller. For each sample, ~12,000 cells were loaded for capture onto the Chromium System using the v2 single cell reagent kit (10X Genomics). Following capture and lysis, cDNA was synthesized and amplified (12 cycles) as per manufacturer's protocol (10X Genomics). Amplified cDNA was used to construct a sequencing library according to the manufacturer's instructions (Illumina).
Experimental Factor: compound
saline
Experimental Factor: dose
1.5