GSM6031815: 5310-WT-B-p53; Mus musculus; RNA-Seq
Tumors were dissected from the brain, minced into pieces < 1 mm3, and digested with an enzymatic mixture that includes papain (0.94 mg/ml, Worthington, LS003120), EDTA (0.18 mg/ml, Sigma, E6758), cysteine (0.18 mg/ml, Sigma, A8199), and DNase I (60 μg/ml, Roche, 11284932001) in 2 ml HBSS (Gibco, 14175-095). Tumor tissues were kept at 37°C for 15 minutes with occasional agitation. The digestion was terminated with the addition of 2 ml Ovomucoid (0.7 mg/ml, Worthington, LS003086). Following digestion, single cells were passed through a 70 μm cell strainer, centrifuged, and resuspended in 30% Percoll (GE Healthcare, 17-0891-01) solution containing 10% FBS (Hyclone SH30396.03). Cells were separated by centrifugation at 800g for 15 minutes at 4°C. The pellet was resuspended in RBC lysis buffer (BioLegend, 420301) for 1 min at 37°C before they were centrifuged at 450g for 5 min. The pellet was resuspend in PBS and immediately dilvered for GEM preparation. Gel-Bead in Emulsions (GEMs) were generated on the sample chip in the Chromium controller. Barcoded cDNA was extracted from the GEMs by Post-GEM RT-cleanup and amplified for 12 cycles. Amplified cDNA was fragmented and subjected to end-repair, poly A-tailing, adapter ligation, and 10X-specific sample indexing following the manufacturer's protocol. Libraries were quantified using Bioanalyzer (Agilent) and QuBit (ThermoFisher) analyses