GSM8827027: shCdkn2a_shSlc39a9 #1_treated with IgG; Mus musculus; RNA-Seq
To obtain a single-cell suspension, dissected tumors were enzymatically and mechanically disrupted. First, the tumors were mechanically minced using surgical disposable scalpels (Surgical Design, catalog no. SCALPEL 12B) into pieces smaller than 5 mm in diameter on a 6 cm plate (Falcon, catalog no. 353002). Next, 2 mL of enzymatic cocktail [8 mg of collagenase IV (Gibco, catalog no. 17104-019), 800 µL of dispase (Stem Cell Technologies, catalog no. 07913), and 25 µg of DNase (Sigma Aldrich, catalog no. D4263-5VL) diluted in FBS-free MEM (Corning, catalog no. 10-009-CV)] was added to the minced tumors. The mixture was incubated for 20 minutes at 37°C in a non-CO₂ incubator. Minced tumor pieces were then further disrupted by smashing them between two fully frosted cytology slides (Fisherbrand, catalog no. 1255015). The resulting single-cell suspension was filtered through a 70 µm strainer (Fisherbrand, catalog no. 22-363-548) and resuspended in 5 mL of FBS-free media. The cells were centrifuged at 350 g for 5 minutes at 4°C and resuspended in 1x RBC lysis buffer (BD, catalog no. 555899). The RBC lysis reaction was incubated for 2–5 minutes at room temperature, after which the reaction was stopped by adding 5 mL of FBS-free media. The cells were then centrifuged at 350 g for 5 minutes at 4°C, resuspended in DPBS, and were ready for subsequent procedures. Libraries were prepared using the single-cell RNA-Seq Benchtop kit (catalog no. 001-100) following the manufacturer's instructions