GSM9765419: B16_unt_Tumor; Mus musculus; RNA-Seq
At day 12, tumours, TDLNs and non-draining LNs were harvested, single cell suspensions were prepared and stained for extracellular antibodies. Single-cell suspensions of (TD)LNs were prepared by mechanically dispersing the lymph nodes through a 100-μm nylon mesh cell strainer. For tumours, single cells were prepared using the Tumour Dissociation Kit (Miltenyi). Using a FACSAria III (BD Biosciences), cells were pooled from 4 sorted (TD)LN populations that were mixed in equal ratios: i) alive ii) CD45- iii) CD45+CD3+CD44+ iv) combined CD45+CD3-CD11b+ and CD45+CD3-CD11c+ cells. From processed tumours, cells were pooled and mixed from 3 sorted fractions: i) alive ii)CD45+CD3+CD44+ iii) CD45+CD3-CD11b+ or CD45+CD3-CD11c+ cells. Next, pooled cell fractions were encapsulated for cDNA synthesis and barcoded with multiplexed runs on a Chromium X using the GEM-X 3' OCM Chip (On Chip Multiplexing). Sequencing libraries were generated following the instructions of the 1061 GEM-X Universal 3' Gene Expression v4 4-plex user guide (CG000768). Sequencing was performed on a Novaseq 6000 platform (Illumina) using an S2 (100 cycle) flowcell with a 28-10-10-90 cycle setting. The target sequencing depth of each library was > 25.000 reads/cell.