Table 1 Procedures for transcriptome sequencing, data analysis and validation
Experimental procedure | Experimental method or threshold considered |
|---|---|
Extraction of total RNA | Trizol reagent (Invitrogen, CA, USA) |
Quality and purity detection of total RNA | RNA 6000 Nano LabChip Kit (Agilent, CA, USA) with RIN number > 7.0 |
cDNA library construction | The cleaved mRNA fragments were reverse-transcribed to create the final cDNA library by the instructions of the mRNA-Seq sample preparation kit (Illumina, San Diego, USA) |
Sample library construction | Illumina Hiseq 2500 sequencing platform |
Transcript abundance estimation | The RPKM of each gene were calculated by the MARS model in DEGseq program, when the RPKM greater than 1000, it is considered to be a highly expressed gene |
Threshold of differentially expressed genes | FDR (false discovery rate) < 0.001 and |log2fold change| ≥ 1 |
Bioinformatics analysis | GO and KEGG enrichment |
Verification of mRNA expression level | Real-time fluorescence quantitative PCR |
Verification of protein expression level | Western blot |