GSM9229485: Relapse 2; Mus musculus; RNA-Seq
Tumor tissues were harvested from three syngeneic colorectal tumor models. Approximately 10–15 mg of tissue per sample was minced in 2 mL of DMEM containing 10% FBS, ulinastatin (2 μg/mL), and sivelestat (2 μg/mL) on ice. Subsequently, 8 mL of digestion buffer (DMEM supplemented with Collagenase D and DNase) was added, and the mixture was incubated at 37 °C in a water bath with gentle shaking (100 rpm) for 1 hour. Following enzymatic digestion, the suspension was filtered through a 40 μm cell strainer, centrifuged at 380 × g for 5 minutes at 4 °C, and the pellet was resuspended in 1 mL of pre-warmed ACK lysis buffer for 3 minutes at 37 °C. After red blood cell lysis, 9 mL of HBSS was added, followed by a second centrifugation step. The resulting cell pellet was resuspended in 0.05% BSA/PBS and adjusted to a final concentration of 1000 cells/μL in 50 μL for downstream single-cell RNA sequencing. Single-cell libraries were prepared using the 10x Genomics Chromium Single Cell 5′ Gene Expression kit according to the manufacturer's protocol.