GSM9318434: Patient 2, GBM, Sample 2, Enhancing; Homo sapiens; RNA-Seq
Human tumor samples were dissociated into single cells utilizing Accutase dissociation reagent (Gibco, NH). Cells were passed through a 70µm strainer (Alkali Inc), centrifuged, and resuspended followed by application of RBC lysis buffer (Biolegend). Single cell suspensions were subjected to counting on the LUNA Fx7 Automated Cell Counter (Logos Biosystems) and diluted to a concentration of 1000 cells/µL. Single cell 3' gene expression libraries were generated using the 10x Genomics Chromium instrument following the manufacturer's protocol. In brief, suspensions were loaded onto the 10x chip along with reverse transcription (RT) master mix and appropriate gel beads. Following generation of single-cell gel bead-in-emulsions (GEMs), reverse transcription was performed, and the resulting Post GEM-RT product was cleaned up and the cDNA amplified. cDNA was subjected to enzymatic fragmentation and size selection to optimize the cDNA size prior to final library construction following the manufacturer's protocol (10x Genomics; 3' v3 (patient). Final library quality was assessed using the LabChip GX (PerkinElmer). Pooled libraries were then subjected to paired-end sequencing according to the manufacturer's protocol (Illumina NovaSeq 6000).