RNA-seq analyses of ‘Pinyiensis’ and bioinformatics analysis
Two sets of samples, comprising one treatment (15 mM KNO3) and one control (15 mM KCl), with three biological replicates each were subjected to RNA-seq. Each biological replicate contained 10 seedling samples. Seedlings with the same growth status were pre-treated with 15 mM KCl for 3 days and subsequently, one set of seedlings were treated with 15 mM KNO3 and the other set of seedlings were continue processed with 15 mM KCl (both under low Fe treatment). After 12 h treatment, root samples were collected and frozen in liquid nitrogen, respectively. Samples were ground into powder in mortar and stored at -80℃. Total RNA was extracted using mirVana™ miRNA ISOlation Kit Ambion-1561 (ABI, Carlsbad, USA) following the manufacturer’s protocol. RNA integrity was evaluated using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). The samples with RNA Integrity Number (RIN) ≥ 7 were subjected to the subsequent analysis. The libraries were constructed using TruSeq Stranded mRNA LTSample Prep Kit (Illumina, San Diego, CA, USA) according to the manufacturer’ s instructions. Total RNAs eliminating mRNA were used for strand-specific library construction. Then these libraries were sequenced on the Illumina sequencing platform (HiSeqTM2500) by OE Biotechnology (Shanghai, China). Reads obtained from sequencing were filtered to remove adaptors and low-quality reads using Trimmomatic (Bolger et al. 2014). The clean reads from each sample were mapped to the reference genome with hisat2.