A total amount of 3 μg RNA per sample was used for the library preparation [25]. The NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA) was used to generate sequencing libraries as described by the manufacturer. The mRNA was enriched by Oligo (dT) magnetic beads and the mRNA is randomly interrupted. Using fragmented mRNA as a template, the first strand of cDNA was synthesized in the M-MuLV reverse transcriptase system, and the second strand of cDNA was synthesized using DNA polymerase I. The purified double stranded cDNA was repaired at the end, “A”tail was added and connected to the sequencing adaptor. The cDNA of about 250-300 bp was screened by AMPure XP beads and amplified by PCR. The PCR product was purified by AMPure XP beads again, and finally the library was obtained. The libraries were sequenced on an Illumina Hiseq X ten platform and paired-end reads were generated. Clean reads were obtained by removing reads containing adapter, reads containing ploy-N and low quality reads from raw data. Fragments per kilobase of transcripts per million mapped reads (FPKM) was used to estimate the gene expression level. Differential expression analysis was performed using the DESeq R package (1.10.1). Genes with an adjusted P-value < 0.05 found by DESeq were assigned as differentially expressed [25].

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