title: GSM6047619: Fetus3; Capra hircus; ncRNA-Seq
library_construction_protocol: A total amount of 2.5 ng RNA per sample was used as input material for the RNA sample preparations. Total RNA was isolated from LD muscle samples by TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's procedure. The concentration and quality of RNA were determined by NanoDrop ND-1000 (NanoDrop, Wilmington, DE, USA) and Agilent 2100 with RIN number >7.0. Approximately 5 μg of total RNA was used to remove ribosomal RNA with the Ribo-Zero™ Kit (Illumina, San Diego, USA). Then ribosome-depleted RNA was treated with RNase R (Epicentre Inc, Madison, WI, USA) to remove linear RNAs and to enrich circRNAs. Then the enriched circRNAs were fragmented into small pieces using divalent cations under high temperature. Retrotranscription of cleaved RNA was performed with PrimeScript RT regent Kit (TaKaRa, Japan), 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. Single-or dual-index adapters containing a T-base overhang are ligated to the DNA fragments. The ligated cDNA products were purified with AMPure XP beads (Beckman Coulter, Beverly, MA, USA), and treated with uracil DNA glycosylase to remove the second-strand cDNA. The purified first-strand cDNA was 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 s, and extension at 72℃ for 30 s; and then final extension at 72℃ for 5 min. At last, the qualified library was sequenced using an Illumina NovaseqTM 6000 (Illumina, San Diego, CA, USA) on a 150 bp paired-end run.