GSM9449375: Muscle,case2; Mus musculus; RNA-Seq
The mouse muscle tissue was added to 1 mL of pre-cooled lysis buffer and minced into approximately 1 mm³ pieces, followed by the addition of 2 mL of pre-cooled lysis buffer. The tissue was then transferred to a Kimble Dounce tissue homogenizer placed on ice and gently homogenized 3–5 times by slowly moving the pestle up and down until complete lysis was achieved. The lysate was incubated on ice for 5 minutes. Subsequently, 4 mL of pre-cooled 2% BSA solution was added for dilution, and the reaction was terminated by pipetting with a Pasteur pipette. The suspension was filtered through a 30 μm cell strainer, and the filtrate was collected and centrifuged at 300 g for 5 minutes at 4 °C. The supernatant was discarded, and the pellet was resuspended in 5 mL of 2% BSA solution. Then, the filtrate was centrifuged at 300 g for 5 minutes at 4 °C. After discarding the supernatant, the nuclei were resuspended in 500 μL of resuspension buffer (1× PBS, 2% BSA, 0.1% RNase inhibitor). The nuclei were stained with propidium iodide, and cell debris was removed by sorting using a BD FACSAria II flow cytometer. A clean nuclear suspension was collected, then counted using CountStar. The scRNA-seq libraries were constructed with the Chromium Single Cell 3' Reagent Kits Version 4 (10x Genomics).