Illumina HiSeq 2500 paired end sequencing; Single cell TCR-seq of T cells isolated from MC38 mouse tumors
Single cell TCR-seq of T cells isolated from MC38 mouse tumors
Mouse 2_p
Established MC38 tumors (~190 mm3) were excised and single cell suspensions were stained and sorted for CD45+TCRβ+T cells.   MC38 (diluted in HBSS and matrigel) was inoculated in the right unilateral flank (in the border of positions B2 and B3) of C57BL/6 mice (ref Study 16-3384 AV). Tumors were taken one day after group-out (average 150-250 mm3 at day 0), approximately 14-19 days. Tissues were dissociated and flow sorted accordingly to obtain the following groups for 10x Chromium 5' Gene Expression Profiling. Our results indicate that the degree of clonal expansion is correlated with expression of T cell exhaustion markers, and that T cells with strong exhaustion phenotype also express high levels of activation markers, such as interferon gamma. Sorted cells were loaded onto a 10x Chromium Chip using reagents from the Chromium Single Cell 5' Library and Gel Bead Kit (10x Genomics) according to the manufacturer's protocol.  Amplified cDNA was used for both 5' RNA-seq library generation and TCR V(D)J targeted enrichment using the Chromium Single Cell V(D)J Enrichment Kit for Mouse T Cells (10x Genomics). 5' RNA-seq and TCR V(D)J libraries were prepared following the manufacturer's user guide (10x Genomics). The final libraries were profiled using the Bioanalyzer High Sensitivity DNA Kit (Agilent Technologies) and quantified using the Kapa Library Quantification Kit (Kapa Biosystems).