RNA-seq analysis of tumor-infiltrating effector T cells isolated from subcutaneous mouse Lewis lung carcinoma (LLC) tumors

Mice bearing subcutaneous LLC tumors were injected intravenously with the 2-deoxyglucose analog 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino)-2-deoxyglucose (2-NBDG, Life Technologies, catalog: N13195). 2-NBDG was used at 25 mg/kg of mouse body weight; 3.33 mg/ml in PBS. 2-NBDG was allowed to circulate for 15 min before euthanizing the mice and excising the tumors to isolate effector (CD44+, CD62L-negative T cells) TILs.

The 15% of effector CD4 or CD8 TILs with the highest 2-NBDG staining were sorted as 2-NBDG Hi cells, and the lowest 15% were sorted as 2-NBDG Lo. 4 independent mice were used. For each population, a pool of 2000 cells (500 cells per mouse) was done. RNA of the respective pools was extracted with the RNeasy Microkit (Qiagen, Catalog: 74004), and cDNA libraries were prepared from poly A RNA (mRNA) using the Smart-Seq2 single-cell protocol (Picelli, et al., 2014, Nat.