GSM5515893: SB4 [TCR]; Mus musculus; RNA-Seq
Tumors were harvested 7 days lfollowing MICB induction, single cell suspensions were prepared and stained with antibodies. Briefly, dead cells were excluded by staining with Zombie Green viability dye (Biolegend) and Fc receptors were blocked prior to the addition of PE-conjugated anti-mouse CD45 (clone 30-F11, Biolegend) antibody. 5,000 CD45+ live cells were sorted from each tumor sample using a FACSAria IIIu instrument. Equal numbers of sorted CD45+ cells from 5 mice per group were pooled, pelleted and resuspended at 1x103 cells/µl in PBS containing 0.04% RNase-free BSA. For each of the four groups, 5,000 cells were used for the 10X Genomics 5' v2 single cell assay. Reverse transcription, cDNA amplification and library preparation were performed according to the manufacturer's protocol. Completed libraries were sequenced on the Illumina HiSeq 2500 instrument with rapid-run mode, yielding >40,000 reads per cell.  Amplified cDNA was also used for TCR V(D)J targeted enrichment using the using Chromium Single Cell Mouse TCR Amplification Kit. TCR V(D)J libraries were sequenced on the Illumina HiSeq 2500 platform with a 150bp paired-end configuration.