Novaseq 6 000 platforms

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)].

Total RNA was extracted from three groups of zebrafish embryos at 96 hpf, including control, LPS, and LPS + DSF, using the Trizol reagent (Ambion, Austin, TX, USA) following the manufacturer’s protocol. The RNA was quantified using a NanoDrop 2000 instrument. Libraries were constructed using NEBNext Ultra™ RNA Library Prep Kit for Illumina (NEB, Los Angeles, CA, USA) and subjected to high-throughput sequencing through the Illumina novaseq 6000 platforms (NovoTech, Beijing, China). The clean reads were mapped to the reference genome (Danio rerio: NCBI_GRCz11). Differential expressed genes (DEGs) of the two groups were analyzed using the DESeq2 R package (1.10.1). The false discovery rate (FDR) was used to correct the p value to determine the true DEGs. For analyzing the gene expression difference between two samples, p value < 0.05 and|log2(Fold change)| ≥ 1 were considered significant DEGs and used for further analysis [67 (link)].
Gene ontology (GO) and KEGG pathway examinations were performed on DEGs for enrichment analysis. GO terms and KEGG analysis with corrected p value < 0.05 were considered to be significantly enriched [68 (link)].

^AiPSC, ^ViPSC, ^AiPSC-CM, and ^ViPSC-CM RNA sequencing was performed by Novogene—Advancing Genomics using Illumina NovaSeq 6,000 platforms. RNA quality control was achieved by removing reads containing adapters, having more than 10% of undetermined bases, and low-quality reads. Less than 2% of these reads were removed in all samples. Reads were mapped to human GRCh38 reference genome (Nurk et al., 2022 (link)) using STAR pipeline (Dobin et al., 2013 (link)); the unique mapping rate was above 94% in all samples (Supplementary Table S3). Then, for each gene, the count of transcripts for each sample was recorded.