title: GSM7637532: KSQ-IVP-0027_GP3_Tumor_M6_S6; Mus musculus; OTHER
library_construction_protocol: Cas9-Tg x TCR-Tg OT1 or PMEL CD8 T cells were isolated from freshly harvested mouse spleens and dissociated using a GentleMACS (Miltenyi), and with CD8 T cells purified by negative selection (EasySep Mouse CD8+ T cell isolation kit).  CD8s were then placed in T225 flasks at a concentration of 1M CD8s/ml and activated with CD3/CD28 Dynabeads and 2ng/ml mouse rIL-2.  The following day, T cells were transduced wither either lentivirus (OT1 B16-Ova screen) or retrovirus (PMEL / MC38-gp100 screen).  Dynabeads were removed the following day, with cells resuspended on cRPMI + 32ng/ml IL-2. On Day 4, transduced T cells were harvested and selected by positive selection using either Thy1.1 for OT1s or hCD2 for PMELs. 5x106 Thy1.1+ Cas9-Tg x OT1 CD8 T cells or 7x106 hCD2+ Cas9-Tg x PMEL CD8 T cells were injected into tumor-bearing mice i.v. via tail vein in 200L PBS, with 10x106 CD8+ T cells saved to determine the sgRNA distribution of the input population of T cells.  Group sizes were 7-8 mice per library.  At the indicated time (eg 11-21 days) following adoptive transfer of cells, mice were euthanized and blood was harvested in EDTA tubes, and stored at -80C. Tumors, spleens, tumor draining and non-draining lymph nodes were harvested and processed further for extraction of CD8 T cells using CD8a Microbeads (Miltenyi, cat# 130-049-401) from the spleen and CD45 Microbeads (Miltenyi, cat# 130-052-301) from the tumor. Tumors were digested using the Miltenyi Tumor Dissociation Kit (Cat# 130-096-730) according to the manufacturer's instructions. Genomic DNA was isolated using the Qiamp Blood Midi and Maxi kids, according to the manufacturer's instructions.