GSM5059998: Young_D30_3; Sus scrofa; ncRNA-Seq
Total RNA was isolated and purified using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's procedure. The RNA amount and purity of each sample was quantified using NanoDrop ND-1000 (NanoDrop, Wilmington, DE, USA). The RNA integrity was assessed by Agilent 2100 with RIN number >7.0. Approximately 5 mg of total RNAs was used to deplete ribosomal RNAs according to the manuscript of the Ribo-Zero™ rRNA Removal Kit (Illumina, San Diego, USA). Then left RNAs were treated with Rnase R (Epicentre Inc, Madison, WI, USA) to remove linear RNAs and to enrich circRNAs. After removing ribosomal and linear RNAs, the enriched circRNAs were fragmented into small pieces using divalent cations under high temperature. Then the cleaved RNA fragments were reverse-transcribed to create the cDNA,which were next used to synthesise U-labeled second-stranded DNAs with E. coli DNA polymerase I, RNase H and dUTP.. An A-base is then added to the blunt ends of each strand, preparing them for ligation to the indexed adapters. Each adapter contains a T-base overhang for ligating the adapter to the A-tailed fragmented DNA. Single- or dual-index adapters are ligated to the fragments, and size selection was performed with AMPureXP beads. After the heat-labile UDG enzyme treatment of the U-labeled second-stranded DNAs,The ligated products are amplified with PCR by the following conditions: initial denaturation at 95℃ for 3 min; 8cycles of denaturation at 98℃ for 15 sec, annealing at 60℃ for 15 sec, and extension at 72℃ for 30 sec; and then final extension at 72℃ for 5 min. The average insert size for the final cDNA library was 300 bp (±50 bp). At last, we performed the 150bp paired-end sequencing on an Illumina Hiseq 4000 (LC Bio, China) following the vendor's recommended protocol.