title: scRNA-seq and scTCR-seq of tetramer-positive T cells from IL27 treated tumor bearing mice
design: scRNA-seq and scTCR-seq of tetramer-positive T cells from IL27 treated tumor bearing mice
library_construction_protocol: For M86-specific CD8+ T cell sorting (for TCR/scRNA-seq), cells from tumor and lymph node digestion were enriched for CD8+ T lymphocytes by negative selection with the EasySep mouse CD8+ T cell Isolation Kit (STEMCELL technologies cat. 19853). The enriched lymphocytes were subsequently stained and FACS-sorted for scRNAseq. Cells were labelled with PE-conjugated and barcoded (total-seq C0952, BioLegend) M86 tetramer for 20 min at room temperature, then stained for surface antigens (CD8-FITC, CD45.2 PE-Dazzle, Thy1.2 Alexa-700, CD4 BUV395) plus one of the following Hashtag antibodies (one for each replicate, 5 μg/ml): TotalSeqTM-C0301 , -C0302, C0303 , -C0304, -C0305, -C0306, -C0307, -C0308, -C0309, -C0310. Viable T cells from tumor and dLN were sorted as CD45.2+Thy1.2+CD4−CD8+ Tetramer+. Cells were sorted using a BD FACSAriaTM Fusion flow cytometer (BD Biosciences, San Jose, CA). Two to 50 thousand cells were sorted in 300 μL of PBS + 2% FCS and kept at 4 °C. Cells were then counted and resuspended at an adequate concentration for loading into the 10X chip. Gene expression and TCR libraries were generated using the Chromium Single Cell 5′ Library and V(D)J Reagent Kit (10X Genomics) according to manufacturer's recommendations. 20,000 cells per sample were loaded into each channel of the Chromium Chip, and recommendations were followed assuming targeted cell recovery of 2,000–10,000 cells. 10X Geomics single-cell gene expression libraries were quantified with the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific) and profiled using the Bioanalyzer High Sensitivity DNA Kit (Agilent Technologies).