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OT-1 T cells were isolated from spleen and lymph nodes of OT-1 transgenic mouse and stimulated with recombinant mouse IL-2, mouse IL-7 and mouse IL-15 for two days. For T cell co-culture screen, MC38-OVA cells transduced with mMCE sgRNA library post puromycin selection were seeded with library coverage more than 2000 fold in each T750 flask (total 12 flasks). Next day, effector T cells were planted in each flask with mouse IL-2 added. MC38-OVA cells with library coverage more than 4000 fold were collected as baseline. With no-T cells as the negative control, tumor cells were collected at day 1, day 2 and day 2.5 with library coverage more than 1000 fold post co-culture for NGS as described in the in vivo screen. |
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