GSM9664313: T cells, melanoma, knocked out with the 57-gene panel [HTO]; Mus musculus; RNA-Seq
Naïve CD8 T cells were isolated from spleens of H11-Cas9 × OT-I mice (6–16 weeks old, male and female) using magnetic bead–based enrichment for naïve CD8 T cells. Purified naïve CD8 T cells were transduced with pooled lentivirus on retronectin-coated plates in complete T cell medium supplemented with IL-7 and IL-15 at 10 ng/mL. Viral input was titrated on T cells to achieve 20–40% Thy1.1 positivity, consistent with predominantly single-perturbation transduction in pooled screens. Cells were maintained in IL-7/IL-15–supplemented medium for 5 days post-transduction without TCR activation. On day 5 post-transduction, Thy1.1+ cells were enriched by magnetic selection to generate a transduced donor population. Enriched Thy1.1+ OT-I Cas9+ CD8+ T cells were adoptively transferred into congenic recipient mice by tail vein injection (5 × 105 cells per mouse). Congenic markers varied by experiment; donor and recipient combinations (CD45.1/CD45.2) together with Thy1.1 expression were used to enable identification of transferred T cells by flow cytometry. T cells were transferred one day prior to tumor implantation. Recipient mice were implanted subcutaneously with B16-OVA melanoma cells (5 × 105 cells per injection) in 200 µL PBS into bilateral flanks. Tumor growth and general health were monitored in accordance with animal welfare guidelines. Tumors were excised, minced, and dissociated using Miltenyi tumor dissociation kits to recover tumor-infiltrating lymphocytes. Dissociated material was filtered to remove debris, and lymphocytes were enriched by density gradient separation with LympholyteM (Cedarlane). CD8 T cells were subsequently purified from tumor infiltrates using Miltenyi magnetic bead–based CD8 enrichment. Only tumor-derived T cells were used for downstream single-cell profiling. To enable multiplexing of samples from individual mice in the second validation screen (Screen 2), purified tumor-infiltrating T cells from each mouse were stained with BioLegend mouse TotalSeq-C hashtag antibodies according to the manufacturer's protocol, including stringent washing steps to minimize ambient oligonucleotide background. Hashtag-labeled samples were pooled and processed using the 10x Genomics Chromium 5′ single-cell gene expression workflow with Feature Barcode chemistry for cell-surface antibody–derived tags and direct CRISPR guide capture. Libraries were prepared following the manufacturer's instructions to generate: (i) 5′ gene expression libraries, (ii) feature barcode libraries for hashtags and cell-surface proteins when appropriate, and (iii) guide capture libraries reporting sgRNA identity. Final libraries were sequenced on an Illumina NextSeq platform using 10x Genomics–recommended read configurations and target sequencing depths for 5′ gene expression, feature barcode, and direct guide capture libraries.