GSM6744379: IgG treated tumor- scRNA-seq; Mus musculus; RNA-Seq
Tumors were trimmed into 2 mm3 pieces in dish before digestion (10 ml of solution with 0.1% DNase I and 0.1% collagenase IV). The mixed solution was then incubated at 37°C for 30min with a speed of 120 rounds/min, which was then applied to a gentle MACS dissociator for further decomposition. 10ml of 2% FACS solution was added to stop digestion, and the mixed solution was filtered through a 70-µm mesh. Centrifugation at 1500 rpm was performed to obtain cell pellets, which was separated by Percoll solution at gradient dilutions of 30% and 70%. Anti-CD45 magnetic beads (StemCell) were used to enrich CD45+ lymphocytes, which were then loaded into platform with original cell pellets at the ratio of 1:1, after viability examination over 90%. Library was performed according to the manufacter's instructions (single cell 3' v2 protocol, 10x Genomics). Briefly, GCs 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.