title: RNA-Seq of Anas platyrhynchos: DEF cells
design: After extracted, total RNAs were treated with RNase R to degrade the linear RNAs, and purified using RNeasy MinElute Cleanup KitQiagen. Next, strand-specific library was constructed using VAHTS Total RNA-seq (H/M/R) Library Prep Kit for Illumina following the manufactures instructions. Briefly, ribosome RNAs were removed to retain circRNAs. The enriched circRNAs were fragmented into short fragments by using fragmentation buffer and reverse transcripted into cDNA with random primers. Secondstrand cDNA were synthesized by DNA polymerase I, RNase H, dNTP (dUTP instead of dTTP) and buffer. Next, the cDNA fragments were purified with VAHTSTM DNA Clean Beads, end repaired, poly(A) added, and ligated to Illumina sequencing adapters. Then UNG (Uracil-N-Glycosylase) was used to digest the second-strand cDNA. The digested products were purified with VAHTSTM DNA Clean Beads, PCR amplified, and sequenced using Illumina HiSeqTM 2500