GSM9613869: PTS_HGG_P80; Mus musculus; RNA-Seq
For scRNA seq, tumor-bearing mice were anesthetized under isoflurane inhalation. Tumors were dissected on ice based on the presence of GFP (Leica M80 stereomicroscope). Tumors were dissociated by incubation with papain (Worthington, no. LK003178) and DNaseI (Worthington, no. LK003172). Following the manufacturer's instruction, red blood cells (RBC) were removed using an RBC removal kit (Miltenyi, 130-094-183). Debris was removed using a debris removal kit (Miltenyi, 130-109-398), followed by dead cell removal with the dead cell removal Kit (Miltenyi,130-090-101). For scRNA seq, a single-cell gene expression library was prepared using the Chromium Single Cell Gene Expression 3v3.1 kit (10x Genomics, PN-1000268). In brief, single cells, reverse transcription reagents, gel beads containing barcoded oligonucleotides, and oil were loaded on a Chromium controller to generate single-cell Gel Beads-In-Emulsions (GEMs) where full-length cDNA was synthesized and barcoded for each single cell. Subsequently, the GEMs were broken, and cDNA from each single cell was pooled. Following cleanup using Dynabeads MyOne Silane Beads, cDNA was amplified by PCR. The amplified product is fragmented to optimal size before end-repair, A-tailing, and adaptor ligation. The final library was generated by amplification.