2.6 RNA-seq and data analyses
Total RNA from different tobacco tissues was extracted with Trizol reagent (Invitrogen), and the Agilent BioAnalyzer 2100 was adapted to determine the RNA integrity. Qualified RNA samples were then used for the sequencing library construction with an Illumina TruSeq RNA Sample PreKit, and the sequencing program was performed on the Illumina Hiseq 2500 sequencing platform by Beijing Annoroad Co. Ltd. Data analysis was performed as described in our previous study (Z. Wang et al., 2020). Briefly, the quality of the mRNA-Seq reads was evaluated by the FastQC method (http:// www.bioinformatics.bbsrc.ac.uk/projects/fastqc/). The adaptor sequence and the low quality (<20) bases at the 5’ and 3’ ends were removed by Trimmomatic (v0.30) (Bolger, Lohse, & Usadel, 2014). The clean reads were mapped to the reference genome of N. tabacum(ftp://ftp.solgenomics.net/genomes/Nicotiana_tabacum/) (Edwards et al., 2017) with HISAT2 software (v2.1.0) (Kim, Langmead, & Salzberg, 2015). Gene expression levels were calculated with Cufflinks (v2.2.1) (Trapnell et al., 2010), and Cuffdiff (Trapnell et al., 2013) was used to identify the DEGs (differentially expressed genes) with log2 ratios ≥2 or ≤−2 (FDR<0.05) . All the DEGs are listed in Supplementary Table S4 and Table S5.