title: MEL2, scRNAseq
organism: Homo sapiens
description: Protocols: Humor tumor samples were collected from patients. Resected human tumors were minced and digested using Native Bacillus licheniformis protease (Creative Enzymes, NATE-0633) on ice for 30 minutes to dissociate into single cell suspension. Tumor single cell suspensions with low viability were further processed using dead cell removal kit (Miltenyi, 130-090-101). Cultured cells were collected stained with TotalSeq anti-human hashtags C0251- C0258 (Biolegend), viability dye (zombie red, Biolegend 423109) and anti-human CD45-FITC (clone HI30, Biolegend 304038) and enriched for live cells and up to 50% immune cells using BD FACS Aria II.  Cells were plated and stimulated with 1000U/ml human IFNb (pbl assay science 11415), 500ng/ml poly:IC HMW (invivogen, tlrl-pic) transfected with lipofectamine 2000, 10ug/ml non-transfected poly(I:C) HMW, 2ug/ml SLR14 (acquired from Iwasaki lab), 1ug/ml ADU-S100 (invivogen, tlrl-nacda2r), 10ug/ml anti-human CD279 antibody (Biolegend, 329902), and cultured for up to 48 hours.  Sorted cells were immediately loaded into a 10x Chromium reaction.   Chromium Next GEM Single-Cell 5′ kits v.2 were used to generate GEX, VDJ and Cite-seq libraries according to the manufacturer's protocol.