Lysine-specific histone demethylase 1A (KDM1A/LSD1) inhibition attenuates DNA double strand break repair and augments efficacy of temozolomide in glioblastoma

Patient-derived primary GSCs (GSC082209) were established from discarded specimens obtained from glioblastoma (GBM) patients undergoing surgery at UT Health San Antonio. We examined global transcriptional changes by RNA-sequencing using patient derived glioma stem cells (GSCs) that are transduced with KDM1A gRNA to knockout KDM1A. For KDM1A knockout, Scramble and KDM1A-specific gRNA were transduced into GSCs possessing TET-inducible Cas9. Cas9 stimulation was done by treating GSCs with 50 ng/mL doxycycline for 14 days, after which cells were validated for KDM1A knockout.The RNA was isolated and utilized for RNA-seq analysis. Overall design: KDM1A knockout (KO) GSCs were generated using CRISPR/Cas9 system. Total RNA was isolated using RNeasy mini kit according to the manufacturer's instructions (Qiagen, Valencia, CA). Illumina TruSeq RNA Sample Preparation was performed following the manufacturer's protocol. Samples were run on an Illumina HiSeq 3000 in duplicate. The combined raw reads were aligned to UCSC hg19, and genes were annotated by Tophat. Genes were annotated and quantified by the HTSeq-DESeq pipeline.