GSM8387352: Tumor, G < G, replicate 1; Mus musculus; RNA-Seq
Tumor tissues freshly collected from syngeneic mice were dissociated into single-cell suspensions using the Tumor Dissociation Kit (Catalog No. 130-096-730, Miltenyi Biotec). Colon tissues were dissected, promptly immersed in ice-cold PBS, and kept on ice for subsequent processing. The colon sections were carefully opened, and any feces present were removed. Colonic tissues were then cut into pieces measuring 1–2 cm in length. Lamina propria cells were isolated using the Miltenyi Biotec Lamina Propria Dissociation Kit (Catalog No. 130-097-410), following the guidelines provided by the manufacturer.  Tissues were processed in a gentleMACS Octo Dissociator with Heaters (Miltenyi Biotec), operating under the program settings recommended by the manufacturer for optimal tissue dissociation while preserving cell viability. The resulting cell suspensions were filtered through a 70 μm cell strainer to remove debris and aggregates. Subsequently, red blood cells were lysed using ACK Lysis Buffer (Thermo Fisher Scientific, Catalog No. A1049201) for 5 minutes at room temperature. Lysed suspensions were then neutralized and washed, and cells were resuspended in ice-cold Fetal Bovine Serum (FBS, Catalog No. 16140071, Thermo Fisher Scientific) to protect cell integrity. Cell suspensions were then passed through a 40 μm FlowMi cell strainer (Millipore, Catalog No. BAH136800040) to ensure homogeneity for downstream processing. Single-cell suspensions were freshly processed and labeled with Sytox Green (Thermo Fisher Scientific, Catalog No. S7020) to stain dead cells for exclusion. Live cells were sorted using a FACS Aria I flow cytometer (BD Biosciences), collecting only Sytox Green-negative cells. Sorted cells were then washed with PBS containing 2% FBS (Catalog No. 10270106, Thermo Fisher Scientific) to remove any extracellular RNA. Cell viability and number were assessed via Trypan Blue (Catalog No. T8154, Sigma-Aldrich) exclusion using a hemocytometer. Viable cells were encapsulated into Gel beads-in-emulsion (GEMs) using the Chromium Next GEM Single Cell 5′ Kit v2 (10X Genomics, Catalog No. PN-1000263) and Chromium Next GEM Chip G Single Cell Kit (10X Genomics, Catalog No. PN-1000120), following the manufacturer's protocol. Barcoding, reverse transcription, and cDNA amplification were conducted as per standard procedures. Amplified cDNA was subjected to a quality check using the Bioanalyzer High Sensitivity DNA Analysis Kit (Agilent, Catalog No. 5067-4626). The cDNA libraries were then fragmented, size-selected, and underwent end-repair, A-tailing, adapter ligation, and PCR amplification with unique indexing. The final library was validated for quality and quantified using a Qubit 4 Fluorometer with the dsDNA HS Assay Kit (Thermo Fisher Scientific, Catalog No. Q32851). The libraries were normalized based on molarity, pooled, and sequenced on an Illumina NovaSeq 6000 system (Illumina, Catalog No. 20012866) following the manufacturer's instructions for high-throughput sequencing.