Instrument: Illumina HiSeq 4000
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: PAIRED
Construction protocol: Trizol reagent (Invitrogen, Carlsbad, CA, USA) was used to extract total RNA, according to the manufacturer’s instructions. CircRNA sequencing libraries were generated using the rRNA-depleted and RNase R digested RNAs by NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations. miRNA sequencing libraries were generated using NEBNext® Multiplex Small RNA Library Prep Set for Illumina® (NEB, USA) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample. mRNA sequencing libraries were generated using the rRNA-depleted RNA by NEBNext® UltraTM Directional RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations. RNA libraries were prepared for sequencing using standard Illumina protocols For circRNA library preparation, a total amount of 5 μg RNA per sample was used as input material for RNA sample preparation. Firstly, ribosomal RNAs were depleted by using Epicentre Ribo-zero™ rRNA Removal Kit (Epicentre, USA) to get rRNA-depleted RNAs. rRNA-depleted RNAs were further treated with RNase R (Epicentre, USA) and then were subjected to Trizol extraction. Subsequently, sequencing libraries were generated using the rRNA-depleted and RNase R digested RNAs by NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations. The library preparations were sequenced on an Illumina Hiseq 2500 platform and 125bp paired-end reads were generated. A total of twelve small libraries were constructed using ovaries of virgin queens, normal queens, caged queens and released queens. For miRNA library preparation, a total amount of 3 μg total RNA per sample was used as input material for the small RNA library. Sequencing libraries were generated using NEBNext® Multiplex Small RNA Library Prep Set for Illumina® (NEB, USA) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample. The library preparations were sequenced on an Illumina Hiseq 2500 platform and 50bp single-end reads were generated. A total of twelve small libraries were constructed using ovaries of virgin queens, normal queens, caged queens and released queens. A total amout of 3 μg total RNA per sample was used as input material for the mRNA library. Sequencing libraries were generated using the rRNA-depleted RNA by NEBNext® UltraTM Directional RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations. The libraries were sequenced on an Illumina Hiseq 2000 platform and 100bp paired-end reads were generated. A total of twelve libraries were constructed using ovaries of virgin queens, normal queens, caged queens and released queens.