GSM5898296: P13; Homo sapiens; RNA-Seq
Total RNA extraction from GBM patient tissue was performed with TRIzol reagent (Invitrogen) according to the manufacturer's protocol. RNA samples have been subjected to DNase I treatment using a ready-to-use DNA-free™ DNA Removal Kit reagents following the manufacturer's instructions (Ambion). The quality of purified RNA was measured utilizing NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific) followed by agarose gel electrophoresis. RNA integrity was verified on an Agilent Bioanalyzer 2100 (Agilent Technologies). RNA extraction from blood was performed with TRI LS Reagent® Solution (Thermo Fisher Scientific). Blood was stored in EDTA bloods in 4°C and RNA was extracted within 4 hours from collection. First, blood was diluted with PBS in 1:2 ratio, then adequate TRI LS Reagent® Solution was added. After incubation with chloroform, centrifugation and supernatant collection isopropanol was added and incubation was performed in –20 °C through the night. Finally, after centrifugation and ethanol precipitation depc-treated water was added and the quality of purified RNA was measured utilizing NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific) followed by agarose gel electrophoresis. RNA integrity was verified on an Agilent Bioanalyzer 2100 (Agilent Technologies). 300 ng of total RNA were rRNA depleted using RNase H according to the protocol (http://dx.doi.org/10.1126/science.aam8526). Ribosomal depleted libraries were constructed using Illumina's TruSeq Total RNA Library Prep Kit. Adaptor ligation were performed according to manufacturer's instructions and the cDNA fragments were amplified with qPCR for 8-15 cycles. After the purification with AMPure XP beads the DNA concentration was measured with Qubit and the fragments length were defined with Screen Tape Assay Agilent D1000, 4200 Tape Station System.