Approximately 1 μg total RNA per sample was treated with Ribo-Zero™ Magnetic Kit (Epicentre) to deplete rRNA. First-strand cDNA was generated using random primers reverse transcription, followed by a second-strand cDNA synthesis. The synthesized cDNA was subjected to end-repair and then was 3′ adenylated. Adapters were ligated to the ends of these 3′ adenylated cDNA fragments. Several rounds of PCR amplification with PCR Primer Cocktail and PCR Master Mix are performed to enrich the cDNA fragments. Then the PCR products are purified with Ampure XP Beads. The final library was quality and quantitated in two methods: checking the distribution of the fragment size using the Agilent 2100 Bioanalyzer and quantifying the library using real-time quantitative PCR (qPCR) (TaqMan Probe). The qualified libraries were sequenced pair end on the Hiseq 4000 platform (BGI-Shenzhen, China).

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