GSM8493801: mT4 cells treated with IgG control, scRNAseq; Mus musculus; RNA-Seq
6-8 tumors per treatment group were harvested, minced and digested in collagenase digest buffer (RPMI, Type I collagenase 0.1mg/ml, Type IV collagenase 0.1mg/ml, Dnase 2ul/ml) for 30 minutes at 37°C. Tissue digest was filtered using a 40um mesh filter and was quenched by washing with 2% FBS in PBS. Cells were counted and depleted of dying cells twice using the Dead Cell Removal kit from Miltenyi per manufacturer protocol. CD45 isolation kit from Miltenyi was used to seperate CD45- and CD45+ cells. Equal amounts of CD45- and CD45+ cells were mixed together in single cell suspension and counted once more. Samples were diluted to a final concentration of 700-1000cells/ul. Samples with greater then 85% viability were processed for sequencing. Single cell RNA-sequencing was performed at the University of Michigan Advanced Genomics Core Research Facility. Single-cell 3' library generation was performed on the 10x Genomics Chromium Controller following the manufacturers protocol for the 3' v3.1 chemistry with NextGEM Chip G reagents (10x Genomics, Pleasanton, CA, USA). Library qualtiy was assessed by LabChip GXII HT (PerkinElmer) and wuantified by Qubit (Thermo Fisher Scientific).