RNA-Seq of A2780
Total RNA was extracted from A2780 cells using the TRIzol reagent kit (Invitrogen; Carlsbad, CA, USA) according to the manufacturers protocol. The purity and concentration of the extracted RNA were evaluated by the NanoDrop 2000 optical test. RNA quality was assessed on an Agilent 2100 Bioanalyzer (Agilent Technologies; Palo Alto, CA, USA) and checked by RNase-free agarose gel electrophoresis. All samples were qualified. Full transcription group sequencing technology was used to build two sequencing libraries, the small RNA (sRNA) library and a chain-specific library, with the NucleoScan RNA removed. The miRNA sequence information is obtained from the sRNA library. The small RNA Sample Pre Kit was used to build a library; T4 RNA ligase 2 (truncated) was used to ligate the sRNA 3 and 5 ends. The chain-specific library of rRNA was removed to obtain mRNA and circRNA sequence information. An Epicentre Ribo-Zero kit was used to remove ribosomal RNA (rRNA). Complementary DNA (cDNA) was synthesized by adding fragmentation buffer to randomly break the rRNA-depleted RNAs, which were then used as templates to synthesize the first strand of cDNA using random hexamer primers; the second chain of cDNA was then synthesized. Next, the cDNA fragments were purified using VAHTS DNA Clean Beads (Vazyme; Nanjing, China), end-repaired, added to poly(A) sequences, and ligated to Illumina sequencing adapters. Uracil-N-glycosylase was used to digest the second-strand cDNA. The digested products were purified using VAHTS DNA Clean Beads, amplified using PCR, and sequenced using an Illumina HiSeq 2500 (Gene Denovo Biotechnology Co.; Guangzhou, China).