title: GSM6918454: KPP-EGP Pre-Implantation - GEX [SAM24405162]; Mus musculus; RNA-Seq
library_construction_protocol: FOXN1-deficient nude mice with adoptively transferred tdTomato+ CD3+ T cells bearing KPP-EGFP STAMP microtumors were biopsied 8 days post tumor implantation using a 1 mm Miltex sterile disposable biopsy punch with plunger (Integra Biosciences, NH, USA). 3-6 pooled tissue biopsies were moved into a precooled 1.5 mL tube (Eppendorf, Germany) containing 300µL digestion cocktail consisting of Gibco RPMI 1640 (Thermo Fisher Scientific, MA, USA), 0.1% Gibco fetal calf serum (Thermo Fisher Scientific, MA, USA), 0.1mg/ml Liberase TM (Roche, Switzerland), 0.1mg/ml DNAse I (Roche, Switzerland), 32µM Gibco Actinomycin D (Thermo Fisher Scientific, MA, USA). Tissues were incubated for 30 min at 37°C and 950x RPM on a Thermoblock (Eppendorf, Germany) and mechanically dissociated every 10 min with a pipette. To quench the digestion, the cell suspension was filtered through a 40 µm mesh into pre-cooled FACS filter tube containing quenching buffer of Gibco fetal calf serum (Thermo Fisher Scientific, MA, USA) with 32 µM Gibco Actinomycin D (Thermo Fisher Scientific, MA, USA). The cell suspension was centrifuged at 350xg for 8 min and resuspended in 400 µL Gibco RPMI 1640 (Thermo Fisher Scientific, MA, USA) with 5 µM CalceinBlue (Invitrogen, CA, USA) and a 1:200 dilution of Molecular Probes Fixable Live/Dead Near-IR Dead Cell Stain Kit (Thermo Fisher Scientific, MA, USA). FACS was performed to isolate Calcein Blue-positive and L/D NearIR-negative cells into a precooled 1.5ml Eppendorf tube containing 750µL collection buffer consisting of Gibco RPMI 1640 (Thermo Fisher Scientific, MA, USA), 10% Gibco fetal calf serum (Thermo Fisher Scientific, MA, USA), 32 µM Gibco Actinomycin D (Thermo Fisher Scientific, MA, USA). The cell number and viability were determined on a Vi-Cell XR cell viability analyzer (Beckman Coulter, CA, USA) and scRNAseq library preparation was performed using dual single cell mouse kit 5'/TCR according to the manufacturer's instructions (10x Genomics, CA, USA). scRNA Seq HTO multiplex for SAM24405163  with Antibody counts in LIB5446558_SAM24405163 (B0301 for Desert, B0303 for Inflamed, B0302 for Excluded) and Gene expression in LIB5446560_SAM24405163; SAM24405162 Contains gene expression data only