GSM8003730: Mouse 11, scRNAseq, Gene expression; Mus musculus; RNA-Seq
0.5g sections of each tumor were minced into small pieces and digested in 10 mL RPMI containing 2.5% FBS, collagenase IV (1 mg/ml, Sigma), and DNase I (200ug/ml, Sigma) in 37°C water bath for 45 minutes. These were then strained to obtain a single cell suspension, and cell viability >90% was determined by trypan blue exclusion. The single cell suspension was stained with anti-CD45 FITC at 4°C for 30min after the FcR was blocked with anti-mouse CD16/CD32 at 4°C for 10min, and then the cells were washed three times with 3.5 mL of cell staining buffer and centrifuged for 5 minutes at 400g at 4°C. 1.8x10e5 - 6x10e5 sorted CD45+ cells from each sample were diluted as necessary for appropriate input into the 10x Chromium chip after cell concentration was verified by manual counting with a hemocytometer. Library was performed according to the manufacter's instructions (single cell 5' v2 protocol, 10x Genomics). Briefly, cells were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.