Widespread and Functional Circular RNA in Glioblastoma

Total RNA was extracted using Trizol reagent kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. RNA quality was assessed on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) and checked using RNase free agarose gel electrophoresis. Next, strand-specific rRNA depleted RNA-seq library was constructed using VAHTS Total RNA-seq (H/M/R) Library Prep Kit (Vazyme, Nanjing, China). Briefly, rRNAs were removed and the retained RNAs were fragmented into short fragments by using fragmentation buffer and reverse transcribed into cDNA with random primers. Second-strand cDNA were synthesized by DNA polymerase I, RNase H, dNTP (dUTP instead of dTTP) and buffer. Next, the cDNA fragments were purified with VAHTSTM DNA Clean Beads, end repaired, poly(A) added, and ligated to Illumina sequencing adapters. Then UNG (Uracil-N-Glycosylase) was used to digest the second-strand cDNA. The digested products were purified with VAHTSTM DNA Clean Beads, PCR amplified to complete library construction. To enhance sensitivity of low abundance CircRNA detection, linear RNAs depleted RNA-seq library were also constructed for each sample. total RNAs were treated with RNase R to degrade the linear RNAs, and purified using RNeasy MinElute Cleanup Kit (Qiagen,Venlo,The Netherlands). The following approach was the same to the procedure of strand-specific rRNA-depleted RNA-seq library as above mentioned. All libraries were sequenced using Illumina X10 by Gene Denovo Biotechnology Co. (Guangzhou, China). Raw reads were filtered by fastp(version 0.18.0) to obtain high quality clean reads. Bowtie2 (version 2.2.8) was used for mapping reads to ribosome RNA (rRNA) database. The rRNA mapped reads were then removed. The remaining reads were further used in CircRNA and host gene analysis.