title: Illumina HiSeq 4000 paired end sequencing; RNA-seq of wild-type and transformed Ink4a/Arf-/-; EGFRvIII neural stem cells in separate or co-culture conditions
design: RNA-seq of wild-type and transformed Ink4a/Arf-/-; EGFRvIII neural stem cells in separate or co-culture conditions
library_construction_protocol: Neural stem cells were sorted by FACS and recovered into growth media. Cells were then pelleted and lysed in RLT Buffer. Cells were cultured adherently in standard serum-free NSC growth media in the presence of 20ng/ml EGF and 10ng/ml FGF2. Cells were cultured either separately or in co-culture prior to harvesting and sorting by FACS. RNeasy extraction kit with on-column DNase digestion mRNA was isolated from total RNA using oligo dT beads. After fragmentation the sample was converted to double stranded DNA and ligated to Illumina adapters. The second strand cDNA was then degraded by the UNG enzyme providing directional information to the library. Indexing of each sample was performed by PCR.