GSM5226619: Lmgp66_TCR_1; Mus musculus; OTHER
Splenic CD4+ T cells were enriched with the CD4 T cell negative isolation kit (Miltenyi). Cells were incubated with anti-CD16/32 in sorting buffer (2% FBS in PBS) for 10 min at 4°C to block binding to Fc receptors. Extracellular antigens were stained for 30 min at 4°C in sorting buffer. Cells were washed and resuspended in sorting buffer with SYTOX blue (Invitrogen) for exclusion of dead cells. Cells were sorted into cRPMI, pelleted and resuspended in RPMI-2% FBS. The TCR libraries were prepared following the Chromium Single Cell Immune Profiling Solution protocol. Sorted gp66:I-Ab tetramer-positive cells (>80% viability) were encapsulated at a final concentration of ~120 cells/uL. After the reverse transcription step the barcoded-cDNA was released from droplets and purified with DynaBeads, followed by 14 PCR cycles (98°C for 45 s; [98°C for 20 s, 67°C for 30 s, 72°C for 1 min] x 14; 72°C for 1 min). The resulting cDNA library was used to construct single cell 5` gene expression and TCR VDJ enriched libraries. The library was fragmented, double-size selected with SPRI beads (avg. size 450 bp), reamplified and sequenced on Illumina NextSeq platform (R1 – 26 cycles, R2 – 98 cycles, i7 – 8 cycles) at depth of ~50.000 reads per cell. To construct TCR libraries, 10 ng of barcoded material was amplified by 2-step nested-PCR (10-cycles of PCR (98°C for 45 s; [98°C for 20 s, 67°C for 30 s, 72°C for 1 min] x 10; 72°C for 1 min) followed by an additional 10 cycles of PCR (98°C for 45 s; [98°C for 20 s, 67°C for 30 s, 72°C for 1 min] x 10; 72°C for 1 min) using DNA primers provided in the kit. VDJ region-enriched libraries with average size of 600 bp were sequenced on an Illumina HiSeq 2500 instrument (R1 – 150 cycles, R2 – 150 cycles, i7 – 8 cycles) to obtain ~5000 reads per cell.