title: GSM9206714: MMTV-R26Met syngeneic graft tumor S34; Mus musculus; RNA-Seq
library_construction_protocol: MMTV-R26Met syngeneic tumors were dissected at passage 7 and minced into small fragments, followed by enzymatic digestion for 20-35 min at 37°C. The digestion solution consisted of type IV collagenase (2 mg/ml, Sigma), hyaluronidase (1 mg/ml, Sigma), and DNase I (20 µg/ml, Roche) in DMEM/F12 (Dulbecco's modified Eagle's media/F12, 1/1, ThermoFisher Scientific) supplemented with 10% fetal bovine serum (FBS, Sigma) and penicillin–streptomycin (P/S, 100 U/ml / 0.1 mg/ml, ThermoFisher Scientific). After digestion, samples were centrifuged, and the pellet was incubated on ice for 1 min with red blood cell lysis buffer (ThermoFisher Scientific). They were then washed in PBS and further incubated for 5 min at 37°C with trypsin (0.05%, Gibco). Subsequently, the cells were resuspended in DMEM/F12 + 10% FBS + P/S, and filtered through a 40µm nylon cell strainer to remove aggregates. Cell counting and viability assessment using trypan blue revealed that all samples exhibited viability above 80%, so no sorting was performed. The cell concentration was then adjusted to approximately 1,000 cells/µL for library preparation. Single-cell RNA sequencing libraries were generated using the 10x Genomics Chromium Single Cell 3' Library and Gel Bead Kit v3.1, following the manufacturer's protocol. Specifically, 10,000 cells per sample were loaded onto a Chromium Controller to encapsulate individual cells into Gel Beads in Emulsion (GEMs). After cell lysis within the GEMs, mRNA was reverse transcribed to produce cDNA, which was then amplified by PCR. This cDNA was subsequently used for sequencing library preparation. The libraries were sequenced on an Illumina NextSeq 500 and NextSeq 2000 sequencers, targeting a depth of approximately 60,000 reads per cell. The library generation, validation, and sequencing were performed at the "Transcriptomique et Génomique Marseille Luminy (TGML)" platform.