GSM7349286: day6_KO; Mus musculus; RNA-Seq
Mice were euthanized and dissected at different days after tumor implantation to collect tumor tissues. Tumor tissues were cut into pieces and enzymatically digested with 1 mg/mL collagenase I, 1 mg/mL collagenase IV, and 300 μg/mL DNase I at 37 °C for 45 min. The suspension was then filtered through a 70 μm Nylon Cell Strainer (Corning Life Sciences) and centrifuged at 500 g for 5 min. Erythrocytes were removed by hypotonic lysis for 5 min at room temperature.  The single cells were then incubated for 30 min with FcR blocking anti-mouse CD16/32 antibody followed by incubation with PE anti-mouse CD45 antibody for another 30 min on ice. The CD45+ immune cells were sorted by SONY MA900 Multi-Application Cell Sorter. Library was performed according to the manufacter's instructions (single cell 3' v2 protocol, 10x Genomics). Briefly, sorted immune 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.