circRNA Sequencing Protocol and Analysis

The circRNA sequencing data were retrieved from Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo) (GSE181523 [19 (link)]). The RNA-seq between circPCNXL2 overexpression and control groups is conducted by Biomarker (China). Total RNA extraction was conducted by TRlzol Reagent (Life technologies, USA). Then, we utilized the Ultima Dual-mode mRNA Library Prep Kit for Illumina (Yeasen Biotechnology, China) to generate sequencing libraries. Library sequencing was performed on Illumina NovaSeq6000 platforms (Illumina, USA). Reads containing adapters, poly-N, and low-quality reads in the raw data were removed. The FASTQ reads were aligned to the GRCh38 reference human genome. Differential expression analysis was carried out by DEseq2 R package in R 4.2.1 with R Studio [20 (link)].