GSM4476365: NRAS_INK4A_LN; Mus musculus; RNA-Seq
Axillary lymph node was isolated from Tyr::NRASQ61K;Ink4a−/− mouse after spontaneous development of melanoma. Tissue was dissected into small pieces and digested using collagenase I & IV (5mg/ml each, Sigma Aldrich) for 20 minutes at 37oC followed by 0.05% Trypsin-EDTA (ThermoFisher Scientific) for 5 minutes at RT. Single cells were separated from remaining tissue using 40 μm Falcon Cell Strainers (Corning) and resuspended in 0.01% BSA. Then, cells were fixed in ice-cold methanol for 15 minutes. Cells were washed with PBS and resuspended in PBS containing 0.01% BSA. We loaded ~8,700 methanol-fixed cells into one channel of the Chromium system using the 3' v2 single cell reagent kit (10X Genomics). Following capture and lysis, we synthesized cDNA and amplified for 12 cycles. The amplified cDNA was used to construct Illumina sequencing libraries that were each sequenced on an Illumina NovaSeq6000 system.