GSM8880298: Heart, heart failure; Mus musculus; RNA-Seq
Type II collagenase (Worthington, CA, USA) (13.5 g) and DNase I (Qiagen) (2 mg) were dissolved in 30 mL of F12Ham medium (Thermo Fisher Scientific, MA, USA). The heart was minced using scissors in an ice-cold Petri dish and mixed with a collagenase solution. The solution was poured into an Erlenmeyer flask soaked in a 38 °C warm bath with a magnetic stirrer for 30 min. After a 30-min incubation, the mixture was repeatedly cannulated using an 18G needle with a 10 mL syringe. After cannulation, a 30-minute reaction was performed. Ice-cold PBS (20 mL PBS to stop the reaction. This solution was centrifuged at 450 × g and 4 ℃. The supernatant was removed, and 5 mL of 1X RBC Lysis Buffer (Thermo Fisher Scientific, MA, USA) was added on ice with gentle shaking. Ice cold PBS (45 mL) was added and the mixture was passed through 70-µm and 40-µm filters (pluriSelect, Leipzig, Germany) consecutively. The solution was centrifuged as previously described and the supernatant was removed. CellCover (Anacyte Laboratories, Hamburg, Germany) (3 mL) was added on ice for 5 min. Ice-cold PBS (47 mL) was added and centrifuged as described previously. The supernatant was removed, and 4 mL of ice-cold PBS was added. This solution and Percoll was stacked in the tube, and was centrifuged for 25 minutes at 3000 × g and 25 ℃. The debris layer and Percoll were swiftly aspirated. The cell layer were washed by ice-cold PBS up to 50 mL and centrifuged for 6 min at 450 × g and 4 ℃. The precipitate was washed again in the same manner. Finally, 1mL of CellCover was added before application to the single-cell analysis apparatus. Libraries were constructed using a commercial microdroplet-based platform, Chromium Single Cell 3' v2 (10×Genomics) according to the manufacturer's instructions.