EGF-induced Transcriptome Changes in GBM stem-like cells
GBM neurosphere line GBM1B were depleted of growth factors for 16 hours followed by EGF stimulation for 4 and 24 hours. RNA-seq was used to identify differentially-expressed (DE) genes after EGF treatment. Overall design: Total RNA was extracted using the RNeasy kit (Qiagen). 4 µg of total RNA was subjected to library preparation using Illumina TrueSeq RNA Sample Preparation kit v2 (Illumina). Libraries were assessed using an Agilent Bioanalyzer (Agilent Technologies) to confirm that cDNA fragment sizes were between 200 and 500 bp in length. Sequencing was performed using an Illumina HiSeq 2500 platform (Illumina). The raw reads were aligned to reference human genome build hg19 using HISAT2 with default parameters. For each gene, the number of reads aligned to its exons were counted and summarized into gene level counts by StringTie based on the GENCODE hg19 annotation. Normalization between samples was carried out by R package edgeR, which controls sequencing depth and RNA composition effects.