Total RNA was extracted from six samples using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s procedure. The purity, quantity and integrity (28S/18S) of total RNA were examined by 1.5% agarose gel electrophoresis stained with Goldview nucleic acid dye (Solarbio, Beijing, China), NanoDrop ND-1000 instrument (NanoDrop, Wilmington, DE, USA) and Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). Approximately 1 µg of total RNA with an RNA integrity number (RIN) ≥ 7 was selected for small RNA library construction using a TruSeq small RNA Sample Prep Kit (Illumina, San Diego, CA, USA) following the manufacturer’s protocol. Then six libraries (three WR_S and three YR_S) were sequenced by Illumina Hiseq 2500, and 50 bp single-end reads were generated.
For mRNA, lncRNA and circRNA sequencing, about 5 μg of total RNA from each sample was used to deplete ribosomal RNA using the Ribo-Zero rRNA Removal Kit (Illumina, San Diego, USA) according to the manufacturer’s instruction. After removing ribosomal RNAs, RNAs were fragmented by divalent cations under a high temperature and reverse transcribed into cDNA, which was used to synthesize U-labeled second-stranded DNAs with DNA polymerase I, RNase H and dUTP and buffer. Finally, 150 bp paired-end reads were generated by an Illumina Hiseq 4000 platform based on the paired-end sequencing.
For mRNA, lncRNA and circRNA sequencing, about 5 μg of total RNA from each sample was used to deplete ribosomal RNA using the Ribo-Zero rRNA Removal Kit (Illumina, San Diego, USA) according to the manufacturer’s instruction. After removing ribosomal RNAs, RNAs were fragmented by divalent cations under a high temperature and reverse transcribed into cDNA, which was used to synthesize U-labeled second-stranded DNAs with DNA polymerase I, RNase H and dUTP and buffer. Finally, 150 bp paired-end reads were generated by an Illumina Hiseq 4000 platform based on the paired-end sequencing.