GSM7846769: H3K27M mutant diffuse midline glioma (BG), scRNA-seq; Homo sapiens; RNA-Seq
Tumor samples were separated into single cell suspensions using a combination of mechanical dissociation and enzymatic degradation of the extracellular matrix. Enzymatic digestion of the samples was performed using a Tumor Dissociation Kit (Miltenyi Biotec, Gladbach, Germany) following the manufacturer's instructions. In short, tissues were minced and mixed with 200 μL enzyme H, 25 μL enzyme A, 100 μL enzyme R, and 4.7 mL Dulbecco's Modified Essential Medium and loaded into MACS C Tube (130-094-392, Miltenyi Biotec). It was then dissociated three times using mild MACS™ dissociation reagent (130-093-235, Miltenyi Biotec) and the samples were incubated at 37 °C for 30 minutes between each dissociation step. Large particles, erythrocytes, and dead cells were removed using a filter (40 μm), Red Blood Cell Lysis Solution (130-094-183, Miltenyi Biotec), and Dead Cell Removal Kit (Miltenyi Biotec), respectively. A Chromium Single Cell 3' Reagent kit (10× Genomics, Pleasanton, CA, USA) was used to create scRNA-seq libraries. The suspension of single cells was loaded into the Chromium Single Cell Controller Instrument (10× Genomics) to generate Gel Beads-in-emulsion (GEMs). Then, barcoded full-length cDNA was then used for the reverse transcription reactions, followed by the disruption of emulsions using the recovery agent. DynaBeadsMyone Silane Beads (Thermo Fisher Scientific, Waltham, MA, USA) were used to clean the cDNA. Subsequently, the amplified cDNA was fragmented, end-repaired, A-tailed, index adapter ligated, and library amplified. Qubit dsDNA HS Assay Kit (Q32854, Agilent, USA) was used for library quantification, and the standard of charge was about 400-500bp for the main peak.