GSM9325662: Patient-derived xenograft, GBM12 Tumor; Homo sapiens; Mus musculus; RNA-Seq
Intracranial syngeneic TRP GBM tumors and patient-derived GBM tumors (GBM12, GBM38, and HF2303) were harvested and dissociated using Collagenase Type II (Invitrogen, 17101015) with rotation at 37°C. The cell suspension was passed through a 40um strainer (Fisherbrand, 22363547), 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' v4 (mouse)).  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). Bcl2fastq2 Conversion Software (Illumina) was used to generate de-multiplexed fastq files and the Cell Ranger Pipeline (10x Genomics) was used to align reads and generate count matrices.