title: GSM8453646: Wild type, replicate 3, scRNAseq; Mus musculus; RNA-Seq
library_construction_protocol: Single cells were isolated from mouse tumor tissues following a previously established method. Initially, a 10x digestion buffer was prepared in PBS, containing collagenase type I (450 units/ml, Worthington Biochemical), collagenase type II (150 units/ml, Worthington Biochemical), collagenase type III (450 units/ml, Worthington Biochemical), collagenase type IV (450 units/ml, Thermo Fisher Scientific), elastase (0.8 units/ml, Worthington Biochemical), hyaluronidase (300 units/ml, MilliporeSigma), and DNase type I (250 units/ml, MilliporeSigma). Freshly dissected tumor tissues were cut into 1-2 mm³ pieces using a sterile blade, washed with PBS, and then incubated in 50 mL of 1x digestion buffer on a shaker at 37°C for 45 minutes. The resulting cell suspension was filtered through a 40 μm mesh (MilliporeSigma) and centrifuged at 400 g for 5 minutes. After removing the supernatant, the cell pellet was resuspended in ACK lysing buffer (Thermo Fisher Scientific) and incubated on ice for 5 minutes to lyse red blood cells. Approximately 10,000 cells were used for library construction with the Chromium Next GEM Single Cell 3′ Kit v3.1 (10x Genomics), following the manufacturer's protocol.