title: Illumina NovaSeq 6000 sequencing; IL-33 provision by reprogrammed tumor fibroblastic stromal cells promotes local T cell activity and facilitates melanoma eradication
design: IL-33 provision by reprogrammed tumor fibroblastic stromal cells promotes local T cell activity and facilitates melanoma eradication
library_construction_protocol: Tumors were harvested, collected in RPMI 1640 medium containing 2% FCS, 20mM Hepes (all from Lonza), 1 mg/ml Collagenase P (Roche), 25 μg/ml DNaseI (Applichem) and 0.8U/ml Dispase I (Roche) and dissociated mechanically. Samples were incubated at 37° C for 45 min, with collection of supernatant and resuspension every 15 min. Cell suspensions were filtered and washed in PBS containing 0.5% FCS and 10mM EDTA. Stromal cells were enriched using CD45 and Ter119 MACS microbeads (Miltenyi) to deplete CD45+ hematopoietic cells and Ter119+ erythrocytes.  Mice were subcutaneously inoculated into the flank with 2x10^5 B16F10 melanoma cells. On day 7, mice were treated intratumorally with 1x105 FFU r3LCMV-TRP2 or PBS. Analyses of EYFP+ or PDPN+ fibroblastic stromal cells isolated from the tumors were performed on day 11 (day 4 after treatment). EYFP+ or PDPN+ fibroblastic stromal cells from s.c. implanted B16F10 tumors of r3LCMV-TRP2 or PBS control Cxcl13-Cre/ TdTomato EYFP mice were sorted using the BioRad S3 Cell Sorter. Sorted fibroblasts were run on the 10x Chromium (10x Genomics) system. The cDNA library generation was performed following the established commercial protocol for Chromium Single Cell 3' Reagent Kit (v2 Chemistry)