Hiseq 2000 2 500 4 000 sequencing system

Before preparing the library, use RNeasy Mini Kit (Qiagen) to separate the total RNA according to the manufacturer's instructions. The 2,100 biological analyzer (Agilent Technologies) was used to check the RNA intensity, and only the high quality samples whose RNA Integrity Number value (RIN) was greater than or equal to 6.8 were used to construct the sequencing library. In addition to the superscript III reverse transcriptase (Invitrogen) for the synthesis of the first strand of cDNA, 1 μg total RNA is usually used in conjunction with the TruSeq RNA library preparation kit (Illumina). The concentration of the ligated fragment with the adapter was enriched and purified by PCR, and the DNA concentration with the adapter was determined by quantitative PCR (Applied Biosystems 7,500). The length of DNA fragment was measured by 2,100 biological analyzer, and the insertion size was 200 bp. Then, the RNA sequence library is sequenced using the Illumina HiSeq 2000/2,500/4,000 sequencing system. The library adopts paired end strategy, and the reading length is 101 bp, 125 bp or 150 bp respectively.

TCGA glioblastoma multiforme (GBM) gene expression data by AffyU133a array platform and clinical data were downloaded from UCSC Xena website (https://tcga.xenahubs.net). Data were standardized by “affy RMA” method, then were transformed into log₂- for further analysis. “somatic.maf.varscan” file of GBM was downloaded from TCGA (https://portal.gdc.cancer.gov/) and was used to calculate Tumor Mutation Burden (TMB) score. Data from Chinese Glioma Genome Atlas (CGGA) website (https://www.cgga.org.cn/) were used as test and verification of the findings in mining TCGA [21 (link)–24 (link)]. RNA-seq libraries were sequenced by the Illumina HiSeq 2000/2500/4000 Sequencing System, then FPKM (fragments per kilobase transcriptome per million fragments) method was used to quantify in CGGA part B dataset. Gene expression data from CGGA part C dataset were performed on all samples using the Agilent Whole Human Genome Array and normalized using GeneSpring GX 11.0 software. All patients included in this study were not less than 18 years old.