RNA-seq of Patient-derived GBM  Cells #8
The MGMT mRNA probes labeled with biotin at 3 end were designed and purchased from RiboBio. RNA pulldown assay was performed according to a standard protocol (Torres et al., 2018). GBM cells were treated with or without TMZ (100 M) for 2 hours and fixed with a 1% paraformaldehyde solution in PBS for 10 min at room temperature which then were quenched by adding 1/10 volume of glycine 1.25 M for 5 min. Cells were washed 2 times with PBS and spined at 510 g at 4 C for 5 min to collect the cells. Cell pellets were lysed in 1mL lysis buffer containing 50 mM Tris-HCl (pH=7.0), 10 mM EDTA, 1% SDS, 200 U RNase inhibitor and 5 l/ml cocktail. The cell lysates were sonicated on ice until the length of RNA fragments range between 200 and 800bp and centrifuged at 12,000g for 5 min at 4 C. The supernatant was transferred to a new centrifuge tube and added 2 volumes of hybridization buffer (50 mM Tris-HCl pH 7.0, 750 mM NaCl, 1 mM EDTA, 1% SDS, 15% formamide). 100 pmol of biotinylated MGMT mRNA probes were incubated with the supernatant for 6 hours at room temperature. Samples then were incubated with 50 l magnetic streptavidin beads, 200 U/ml RNase inhibitor and 5 l/ml cocktail overnight at room temperature. The beads were washed 6 times with wash buffer (0.5% SDS and 2x SSC) and then incubated with proteinase K for 45 min at 50 C then 10 min at 95 C. RNAs were purified using RNA purification followed by cDNA synthesis and RNA amplification, according to the manufacturers instructions. The RNA-seq libraries were constructed and sequenced utilizing the Illumina HiSeq2500 platform (Illumina, San Diego, USA).