title: RNA sequencing of Ink4a/Arf knockout natural killer cells and controls
library_construction_protocol: NK cells were isolated from the spleens of Ink4a/Arf KO mice and WT mice by magnetic cell separation using purified antibodies (CD4, CD5, CD8a, CD19, Ly-6G/Ly-6C; TER-119, F4/80, CD3, I-A/I-E) and magnetic beads. Subsequent fluorescence-activated cell sorting for NK1.1+, CD3-, TCRβ- and TCRγδ- cells was performed.  Ex vivo (non-cultured) NK cells were directly lysed in RLT buffer supplemented with β-mercaptoethanol and stored at -80°C. For the short-term cultured (day 11-22) and long-term cultured (day 49-93) samples, the NK cells were cultured in RPMI-1640 supplemented with 10% FBS, 100U/ml penicillin, 100µg/ml streptomycin, 50µg β-mercaptoethanol and 3000U/ml IL-2 at 37°C with 5% CO2. At the respective timepoints cells were harvested, washed, lysed in RLT buffer supplemented with β-mercaptoethanol and stored at -80°C. For the leukemic Ink4a/Arf KO samples, NK cells from the spleen of diseased mice, which were previously injected with in vitro generated Ink4a/Arf KO NK cell lines, were flow-sorted for CD45.2+, NK1.1+, CD3-, TCRβ- and TCRγδ-. After sorting, the cells were also lysed in RLT buffer supplemented with β-mercaptoethanol and stored at -80°C.  For all samples NK cells were pooled from 2-4 mice, except for the leukemic samples, in which NK cell numbers from a single recipient mouse were sufficient. RNA isolation was performed on the same day for all samples using the RNeasy Mini Kit (QIAGEN).  Libraries were constructed using the Smartseq3 method (Michael Hagemann-Jensen, et al.; Nat Biotechnol. 2020 Jun;38(6):708-714)