RNA-sequencing on human UI226 tumorspheres and xenografts

To identify molecular changes following trametinib treatment, we performed RNA-sequencing (RNA-seq) on drug treated UI226 SHH MB tumorspheres as well as on UI226 xenografts. UI226 tumorspheres were plated at 2 × 10⁵ cells/well into each well of a 6-well ultra-low attachment plate. DMSO control or 250 nM of trametinib was added to the tumorspheres and samples were collected after 3 days (N = 4) or 7 days (N = 3–4) for RNA extraction. For the in vivo study, once UI226 xenograft mice reached endpoint, tumors were resected, and human cells were isolated using a brain tumor dissociation kit (Miltenyi Biotec, Bergisch Gladbach, North Rhine-Westphalia, Germany) followed by a mouse cell depletion kit (Miltenyi Biotec) according to manufacturer’s instructions. RNA was extracted from 3 vehicle control endpoint tumors taken at days 61, 63 and 65 and from 3 trametinib treated xenografts at endpoint taken at days 75, 76 and 80. RNA was extracted using the Norgen RNA extraction kit (Norgen Biotek, Thorold, ON, Canada) according to manufacturer’s instructions. Library preparation and RNA-seq were performed by StemCore laboratories at the Ottawa Hospital Research Institute (Ottawa, ON, Canada) as previously described [19, 32]. Libraries were prepared from 500 ng of input total RNA with stranded mRNA library prep kit (Illumina). RNA-seq was performed comparing control and trametinib treated tumor cells from primary xenografts.