GSM5525394: OP8; Homo sapiens; RNA-Seq
Fresh samples were minced, washed with PBS, and dissociated using a Human Tumor Dissociation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) per manufacturer guidelines. Red blood cell lysis was performed with ACK lysis buffer per manufacturer protocol (Thermo Fisher Scientific), followed by dead cell removal using a dead cell removal kit to improve the viability if needed (Miltenyi Biotec, Bergisch Gladbach, Germany). Viability was confirmed to be >80% in all samples based on trypan blue analysis (ThermoFisher Scientific). Cell suspensions were filtered using a 40 μm filter (ThermoFisher Scientific) and dissociated cells were pelleted and re-suspended in AutoMACS Rinsing Solution with 0.5% bovine serum albumin (BSA; Miltenyi Biotech). The single-cell suspension was sorted using human CD45 magnetic MicroBeads (Miltenyi Biotec) to enrich for CD45+ cells. Briefly, 20μl of CD45 MicroBeads per 107 total cells was added to the cell suspension and incubated for 15 minutes at 2-8°C. After incubation, CD45+ and CD45- cells were collected after the cells passed through the magnetic column. The CD45+ and CD45- cell pellets were then obtained after centrifuging at 450g for 5 minutes at 4°C. Samples were processed using the Chromium Single Cell 3′ (v2 Chemistry), and in two cases 5', platform with the target of ∼10,000 cells (10x Genomics, Pleasanton, CA) following manufacturer's instructions. Briefly, cells were added onto a chip to form Gel Bead-in-Emulsion (GEMs) in the Chromium instrument followed by cell lysis, barcoding, fragmentation, adaptor ligation and addition of sample index to the libraries before sequencing.