GSM3937770: scRNA-seq from C57BL/6 mouse 1; Mus musculus; RNA-Seq
The right flank of mice (C57BL/6 or PMEL) was shaved and B16-F10 cells (2.5x10^5) were injected subcutaneously. Tumors were excised 15 days post tumor engraftment, manually dissociated and digested enzymatically with Tumor Dissociation Kit (Miltenyi Biotec). Digested tumors were mashed through 70 µm filters. Hematopoietic cells were further purified using a discontinuous Percoll gradient (GE Healthcare). Cells at the interface were harvested and washed twice before further use. Tumor infiltrating CD8 T cells were sorted by flow cytometry and were used for single-cell RNA-seq. Cellular suspensions were loaded on a Chromium Single Cell Instrument (10x Genomics, Pleasanton, CA) to generate single-cell GEMs. Single-cell RNA-Seq libraries were prepared using Chromium Single Cell 5' Library & Gel Bead Kit (PN-1000006) following the manufacturer's instruction (protocol CG000086 Rev E), and the 10x mouse V(D)J Enrichment Kit (PN-1000071) was used to enrich for TCR sequences, after which V(D)J-enriched libraries were constructed with the Chromium Single Cell 5' Library Construction Kit (PN-1000020). The sequencing libraries were loaded on Illumina NextSeq High Output Flow Cells using read lengths of 26 nt for read1 and 132 nt for read2.