GSM8746699: CON41; Mus musculus; RNA-Seq
Fresh tissue of the diaphragm is collected from C57/BL6 mice. Diaphragm is placed in the sterile dish and washed with cold PBS buffer for multiple times to remove the blood. Tendon was cut and muscular part was kept. Then, the tissue was fully cut into small pieces of about 0.5mm3 with surgical scissors and incubated within digestion solution which containing collagenase P (5 mg/mL final concentration), dispase II (1.2 U/mL final concentration), and CaCl2 (5 mM final concentration) for 45 min at 37°C. Mix the suspension thoroughly every 10 min. The digestion was terminated and filtered through a 40-µm nylon cell strainer. The flow-through was centrifuged at 300x g at room temperature for 10 min. After the precipitation was suspended with appropriate medium, the same volume of red blood cell lysate was added, mixed, for 10 min at 4℃. After washing, the cell precipitation was suspended in the medium of 100μl, and the cell concentration and viability were calculated by Luna cell counter. The freshly prepared single-cell suspension was adjusted to 700-1200 cell/μl according to the 10×Genomics Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 (No. 1000268) Operation manual for computer and library construction. The constructed library was sequenced using Illumina Nova 6000 PE150 platform.