GSM7082598: GBM_CTRL_D32(CTRL6); Homo sapiens; RNA-Seq
GBM samples were pooled so that there were 2-3 chips (~40-60mg) of GBM tissue, per treatment condition, per each of the 4 patients for RNA extraction. TRIzol™ (ThermoFisherScientific) was added to the tissue biopsies and samples were homogenised using a douncer. Chloroform was added 1:5 to the lysate, tubes inverted gently for 15 seconds and then incubated for 3 minutes on ice. Samples were centrifuged at 12,000 x g for 15 minutes at 4°C. Isopropanol was added 1:2 to the upper, colourless, aqueous phase and samples were incubated overnight at -80°C before centrifuging at 12,000 x g for 10 minutes at 4°C. The RNA pellet was washed twice in 75% ethanol before centrifuging for 5 minutes, at 7500 x g at 4°C and the supernatant discarded each time. The RNA pellet was air dried before resuspending in RNase-free water through incubation at 55°C. Total RNA was then analysed to yield the transcriptomic profile of the tissues in the two different conditions (DMSO control and 1uM GSK3368715 treated.) Library preparation and RNA sequencing were done by Novogene Co. Ltd. (Cambridge, UK) using the Illumina NovaSeq 6000 platform at a depth of 34-60 million reads