RNA library construction and RNA-seq sequencing

The Illumina RNA-Seq procedure (Illumina, San Diego, CA, USA) for RNA library construction was conducted by Nanjing Pisenuo Gene Technology (Nanjing, China). The standard for library construction was RNA integrity number (RIN) ≥ 7, 28 S/18 S ratio > 0.7. The HiSeqTM 2,500 sequencer (Illumina, San Diego, CA, USA) was then used for sequencing with a sample load of 1 μg. The sequencing results were compared and annotated with the database as analysis background data, and the screening conditions for differential expression of circRNA in the two cells line were defined as fold changes (FC) ≥ 2 and P < 0.05.

Analysis of GO enrichment and KEGG pathway analysis of genes derived from the differential circRNAUse GO (Gene Ontology) database (http://www.geneontology.org) was used to perform gene function enrichment analysis on genes derived from differential circRNA, then count the numbers of differential genes included in each GO entry were counted, and the hypergeometric distribution test method was used to calculate the significance of the enrichment of differential transcripts in each GO entry. KEGG (Kyoto Encyclopedia of Genes and Genomes) database (http://www.genome.jp/kegg/pathway.html) was used to analyze the signal pathways (Pathway) of the differential genes, and also used of hypergeometric distribution test to calculatethe significances of the enrichment of differential genes in each pathway entry. The above calculations resulted in a value of P that reflected the significance of enrichment. The smaller the P value, the more significant the tendency of differential genes to be enriched in the GO or Pathway entry.