title: GSM1976024: Primary GBM RCAS mouse model cell T1-69; Mus musculus; RNA-Seq
library_construction_protocol: The tumor cells were isolated from primary or recurrent tumors from RCAS-tva mice followed by FACS. Tumor masses, which were pre-determined by MRI, were minced and then incubated at 37°C with PAPAIN in PIPES buffer, resuspending after 20 min. After quenching the digestion with fetal calf serum, the suspension was again disrupted with a pipette and passed through 70um cell filters. Samples were collected immediately by centrifugation, and then they were subjected to myelin removal (Miltenyi Biotec.). Wash the remaining cells by PBS with 2% fetal calf serum, centrifuge at 300g for 5 minutes, discard supernatant and re-suspend the pellet in the volume remaining. Follow antibody and live/dead stainings (Invitrogen) procedures. Cells were counted and one million cells in each sample were subjected to flow assisted cells sorting.  The following antibodies were used for staining: FITC-CD45, PE-CD31, APC-CD140a (PDGFRa), (eBiosciece); Pacific Blue/Mouse Lineage Panel (Biolegend). The data were analyzed by Flowjo software. The analysis was done within single cell gate after the exclusion of debris and dead cells. Tumor cells were isolated from mouse brain tumor tissue samples. Single cells were then captured and mRNA isolated using Clontech's SMARTer chemistry on the Fluidigm C1 Single Cell Auto Prep system. Illumina's Nextera XT kit was used for library preparation. Single cell sequencing were performed on the HiSeq2500 platform with 100 bp single-end sequencing.
attributes.tag: GEO Accession
attributes.value: GSM1976024