circRNA_DAF115-1 (CRX1174164)
Total RNA was extracted using Trizol reagent (Thermo Fisher, 15596018) following the manufacturer's procedure. The RNA quantity and purity were analyzed using the Bioanalyzer 2100 and RNA 6000 Nano LabChip Kit (Agilent, 5067-1511) and required to meet RIN number > 7.0. Approximately 5 ug of total RNA was used to deplete ribosomal RNA according to the manufacturer's procedure of the Ribo-Zero Gold rRNA Removal Kit (Illumina, MRZG12324). After removing ribosomal RNA, the remaining RNAs were treated with Rnase R (Epicentre Inc, Madison, WI) at 37C for 30 minutes to remove linear RNAs and enrich circRNAs. The remaining RNAs were fragmented (NEBNext Magnesium RNA Fragmentation Module, E6150S) using divalent cations under high temperature. Then the cleaved RNA fragments were reverse-transcribed to create the cDNA, which were next used to synthesize U-labeled second-stranded DNAs. Single- or dual-index adapters were ligated to the fragments, and size selection (300-600 bp) was performed with AMPureXP beads. After the heat-labile UDG enzyme (NEB, m0280) treatment of the U-labeled second-stranded DNAs, the ligated products were amplified with PCR. The average insert size for the final cDNA library was 300 +/- 50 bp. Finally, the 2x150 bp paired-end sequencing (PE150) was performed on an Illumina Novaseq 6000 (LC-Bio Technology CO., Ltd., Hangzhou, China) following the manufacturer's protocol.