Instrument: NextSeq 500
Strategy: OTHER
Source: TRANSCRIPTOMIC
Selection: other
Layout: PAIRED
Construction protocol: For each sample, 10μg of total RNA was used for cirRNA-seq library preparation.Total RNA was treated with RQ1 DNase (Progema) to remove genomic DNA. A large proportion of mRNA was captured by oligo dT(25), and remaining RNA was purified with Ampure XP Beads. Ribosomal RNA was depleted away by RiboMinus Kit. Linear RNA (including residual rRNA, residual mRNA, and so on) was digested by RNase R. CircRNA was iron fragmented at 95℃ followed by end repair and 5’ adaptor ligation. Then reverse transcription was performed with RT primer harboring 3’ adaptor sequence and randomized hexamer. The cDNAs were purified and amplified, then PCR products corresponding to 300~500bp were selectively captured and quantified. Libraries were stored at -70 C until used for sequencing. For high-throughput sequencing, the libraries were prepared following the manufacture’s instructions and applied to Illumina NextSeq500 system with 150x2 paired-end type by ABlife. Inc (Wuhan, China).