GSM6217643: Tum-minus; Mus musculus; RNA-Seq
At the indicated time points, CD8+ T cells were isolated from the spleen and the subcutaneous tumor using mouse CD8+ T cell isolation kit (Miltenyi Biotec) and sorted based on expression of the tomato fluorescent protein (tomato positive = Cd137 minus, tomato negative = Cd137 plus) on a FACSaria™ Fusion (BD biosciences). Cells from 7 mice were stained with hashtagging antibodies (BioLegend) in the following way: mouse 1 = ACCCACCAGTAAGAC; mouse 2 = GGTCGAGAGCATTCA; mouse 3 = CTTGCCGCATGTCAT; mouse 4 = AAAGCATTCTTCACG; mouse 5 = CTTTGTCTTTGTGAG; mouse 6 = TATGCTGCCACGGTA, mouse 7 = GAGTCTGCCAGTATC. Hashtagged cells where then pooled to yield 4 cellular suspension corresponding to spleen Cd137 minus, spleen Cd137 plus, tumor Cd137 minus, tumor Cd137 plus. Single-cell libraries (5′ gene expression) were generated using the Chromium Controller Instrument and Chromium Next GEM Single Cell 5' Library & Gel Bead Kit v1.1 according to the manufacturer's protocol (10× Genomics) The libraries were sequenced on a NextSeq 550 (Illumina) in pair-end sequencing 28 bp (read1) × 91 bp (read2) and a single index 8 bp in length.