Gene expression was normalized using the fragments per kilobase of exon per million reads mapped (FPKM), which was calculated using Cuffdiff (v2.1.1) (22 (link)). Cuffdiff provides statistical routines for determining differential expression in the gene expression data using a model based on the contrary binomial distribution (22 (link)). Subsequently, differentially expressed lncRNAs at H2, H8, and H12 compared with H0 were filtered, and lncRNAs with a p-value < 0.05 were assigned as differentially expressed.
To validate the Illumina sequencing data, a total of six differentially expressed lncRNA were randomly selected for the qRT-PCR analysis. First, cDNA was synthetized using the PrimeScript 1st strand cDNA Synthesis Kit (Takara, Japan). Then, specific primers were designed based on their sequences and EF1α was used as the internal control. The qRT-PCR was performed with the CFX96 Real-time Fluorescent quantitative PCR system (Bio-Rad, USA) using TB GreenTM Premix Ex TaqTM II (TaKaRa, Japan). The amplification cycle was as follows: 95°C for 30 s, 40 cycles at 95°C for 5 s, and 60°C for 1 min, followed by a melting curve from 60 to 95°C. Data are shown as means ± SE for three replicates, and statistical analysis was performed using SPSS19.0.
To validate the Illumina sequencing data, a total of six differentially expressed lncRNA were randomly selected for the qRT-PCR analysis. First, cDNA was synthetized using the PrimeScript 1st strand cDNA Synthesis Kit (Takara, Japan). Then, specific primers were designed based on their sequences and EF1α was used as the internal control. The qRT-PCR was performed with the CFX96 Real-time Fluorescent quantitative PCR system (Bio-Rad, USA) using TB GreenTM Premix Ex TaqTM II (TaKaRa, Japan). The amplification cycle was as follows: 95°C for 30 s, 40 cycles at 95°C for 5 s, and 60°C for 1 min, followed by a melting curve from 60 to 95°C. Data are shown as means ± SE for three replicates, and statistical analysis was performed using SPSS19.0.
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