GSM3358042: GBM_TMZ+4Gy_2; Homo sapiens; RNA-Seq
After radiochemotherapy, all 3 slices of each insert were pooled together, transferred to the QIAzol Lysis Reagent (Qiagen) and homogenized by a hollow needle attached to a syringe. The extraction of total RNA was performed using the miRNeasy Mini Kit (Qiagen) according to the manufacturer's protocol. Isolated RNA was diluted in 30 µl of RNAse-free water and stored at -80°C. RNA concentration was determined using the Qubit 2.0 (Life Technologies). The quality of the isolated RNA was determined by the Bioanalyzer 2100 using the RNA 6000 Nano-Kit (Agilent Technologies). RNA was DNase-digested twice using the TURBO DNA free Kit (Ambion®, ThermoFisher Scientific). For library preparation with the Truseq-Stranded Total RNA Sample Prep Kit (Illumina) up to 200 ng RNA per sample were used. An rRNA depletion step was conducted according to the manufacturer's protocol and – depending on the quality of each sample – a fragmentation was done. Every library was equipped with two barcodes to allow multiplexing of the samples. Concentrations were determined using the Qubit DNA Kit and the DNA quality was detected by the Bioanalyzer 2100 (DNA1000 Kit). According to the average size, which is determined by the Bioanalyzer, and the exact concentration of the samples, the molarity of each library was calculated. The samples were sequenced at the HiSeq2500 with 2 x 126 bp paired-end reads. 12 pM of DNA were put on the flowcell using one lane per sample. The number of reads obtained was between 243 and 368 x 106 reads per sample. Sequencing was performed with 2 x 126 bp paired-end reads at a HiSeq2500 (Illumina) with one sample per lane.
GEO
GSM3358042