Illumina NovaSeq 6000 paired end sequencing; RNA-seq of the primary patient-derived GBM cell line G797 following siRNA-mediated knockdown and pcDNA3.1-Neomycin-mediated overexpression of the lncRNA HOXA10-AS
RNA-seq of the primary patient-derived GBM cell line G797 following siRNA-mediated knockdown and pcDNA3.1-Neomycin-mediated overexpression of the lncRNA HOXA10-AS
G797 cells were maintained in serum-free NeuroCult™ NS-A Basal Medium (STEMCELL Technologies Canada Inc) supplemented with N2, B27, EGF (10 ng/ml), and FGF-2 (10 ng/ml). For overexpression, cells were transfected with pcDNA3.1-Neomycin-HOXA10-AS or the control vector pcDNA3.1-Neomycin using Lipofectamine 3000 Reagent (Thermo Fisher Scientific) as per manufacturer's protocol. Two days post transfection, 500ug/ml G418 was added to select cells with stably expressing genes of interest for 2 weeks. Stable cells with overexpression of HOXA10-AS or the vector only were harvested for RNA extraction. Overexpression of HOXA10-AS was confirmed using RT-PCR. For knockdown, transfection of DsiRNAs was performed using the Lipofectamine RNAiMAX Reagent (Thermo Fisher Scientific) as per manufacturer's protocol. Three days post transfection, cells were harvested for RNA extraction. RNAs were extracted from cells using Quick-RNA Microprep Kit (Zymo Research) as per manufacturer's protocol. The quality of RNAs were assessed by Agilent Fragment Analyzer. The high quality RNAs were used to prepare the sequencing libraries.  RNA-seq libraries were prepared using the Illumina TruSeq Stranded mRNA Sample Prep Kit (20020594) as per manufacturer's protocol. The barcoded cDNA libraries were then checked with Agilent Fragment Analyzer for fragment size and quantified with ddPCR (BioRad) using ddPCR™ Supermix for Probes (No dUTP) (BioRad cat#1863023) running in BioRad CFX96 Touch Real-Time PCR Detection System.