RNA-Seq data from human GBM stem-like cells expressing transgenice TGFBR2
RNA-Seq data from human GBM stem-like cells expressing transgenice TGFBR2
Glioma stem cells without or without transgenic TGFBR2 or Oct4/Sox2 were expanded in neurosphere medium and collected for nucleic acid extraction. GBM-derived neurosphere lines (GBM1A and GBM1B) were originally derived and characterized by Vescovi et al.  All GSCs utilized in this study are models of GBM (IDH wild-type). All neurospheres were cultured in serum-free conditions using Stemline(R) Neural Stem Cell Expansion Medium (Sigma Millipore) supplemented with 20 ng/mL epidermal growth factor and 10 ng/mL fibroblast growth factor.   RNA was extracted using Qiagen RNeasy Kit following manufacturer's instructions.  RNA-Seq libraries were constructed from messenger RNA (mRNA) purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers, followed by the second strand cDNA synthesis using deoxyuridine triphosphate (dUTP). The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection. Quantified libraries were pooled and sequenced on Illumina platforms followed by clustering of the index-coded samples according to the manufacturer's instructions. After cluster generation, the library preparations were sequenced on an Illumina platform and paired-end reads were generated. Index of the reference genome (i.e. hg38) was built and reads were aligned to the reference genome using Hisat2 v2.0.5. Differential expression analysis of two conditions/groups (two biological replicates per condition) was performed using the DESeq2 R package (1.20.0).  ATAC-Seq Libraries were constructed using the Buenrostro60 methodology. Briefly, 1.5x105 freshly isolated nuclei were subject to Tn5 trasposition at 37°C for 30 min using the Nexterra workflow from Illumina. Immediately following transposition, DNA was purified using a Qiagen MinElute PCR Purification Kit and the transposed DNA was eluted in 10 μl of Elution Buffer (10 mM Tris buffer, pH 8). The purified DNA was then barcoded and amplified using a total of 16 PCR cycles. Libraries were then subject to paired-end sequencing, alignment, and differential expression analysis by Novogene as described above.