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.