GSM8076307: primary GBM patient 58 tumor core region; Homo sapiens; RNA-Seq
Total RNA was extracted using the RNeasy kit (Qiagen, Hilden, Germany), according to the manufacturer's instructions. RNA purity was determined by assaying 1 µl of total RNA extract on a NanoDrop8000 spectrophotometer. RNA integrity was checked using an Agilent Technologies 2100 Bioanalyzer with an RNA Integrity Number (RIN) value. Total RNA sequencing libraries were prepared according to the manufacturer's instructions (Illumina Truseq Stranded Total RNA Sample prep kit with Ribo-zero Human/mouse/rat).  Total RNA 400ng was subjected to ribosomal RNA depletion, with Ribo-zero Human/mouse/rat reagent using biotinylated probes that selectively bind rRNA species. Following purification, the rRNA-depleted total RNA was fragmented into small pieces using divalent cations under elevated temperature. The cleaved RNA fragments were copied into first strand. cDNA using reverse transcriptase and random primers. This was followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. These cDNA fragments then had the addition of a single 'A' base and subsequent ligation of the adapter. The products were purified and enriched with PCR to create the final cDNA library. The quality of the amplified libraries was verified by automated electrophoresis (Tapestation, Agilent). After QPCR using KAPA SYBR FAST qPCR Master Mix(Kapa Biosystems), we combined libraries that index tagged in equimolar amounts in the pool. RNA sequencing is performed using an Illumina NovaSeq 6000 system following provided protocols for 2x100 sequencing.