title: Comparative RNA-seq analysis in the basal nodes of nitrogen-stressed hexaploid wheat Tad17 triple knock-out mutant (TILLING) and WT segregant plants
design: Comparative RNA-seq analysis in the basal nodes of nitrogen-stressed hexaploid wheat Tad17 triple knock-out mutant (TILLING) and WT segregant plants
library_construction_protocol: In this study, the basal node is defined as the 0.5 cm long section from the base of the main shoot, which includes the shoot apical meristem, lateral buds, and leaf meristems. After removing all roots from the shoot, any outgrown primary tillers were also carefully removed, and a 0.5 cm section of the main shoot base was dissected. Samples were promptly frozen in liquid nitrogen and stored at -80°C until processing. Tissue samples from four plants were pooled per biological replicate. Plants were hydroponically cultivated in a controlled environment chamber utilizing a custom hydroponic system. Each plant was supported by foam plugs atop 1 L black pots containing the nutrient solution. Continuous aeration of the nutrient solution was maintained throughout the experiment via a tubing system connected to an aeration pump. Growing conditions were set at a daytime temperature of 20°C and a nighttime temperature of 10°C, with a 14-hour photoperiod. Lighting was provided by fluorescent bulbs at an intensity of 550 µmol m-2 sec-1. Humidity levels were maintained at 65% during the day and 75% during the night. The composition of the basic nutrient solution was 1.5 mM Ca(NO3)2, 5 mM KNO3, 2 mM NaNO3, 1.5 mM MgSO4, 1 mM phosphate buffer solution (KH2PO4 – K2HPO4, pH 6.0), 50 μM FeNaEDTA, 0.5 μM CuCl2, 20 μM H3BO3, 3.6 μM MnCl2, 0.1 μM Na2MoO4 x 2H2O and 0.77 μΜ ZnCl2. For Low N treatment, plants were supplied with 0.1 mM of N in the form of KNO3. For ionic balance in the low N nutrient solution, Ca(NO3)2 was replaced by CaCl2, KNO3 by KCl2 and NaNO3 by NaCl. 2.35 mM MES monohydrate was added as a buffer and the pH was adjusted to 5.8 with KOH Total RNA was extracted from approximately 100 mg of ground frozen tissue using the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. Elution of the column was performed with 50 μL of RNase-free H2O. To ensure no DNA carryover, DNase treatment was performed using RQ1 RNase-Free DNase (Promega). After DNase treatment, RNA was further purified by phenol/chloroform/isoamyl-alcohol extraction, followed by RNA precipitation using EtOH/NaOAc. Finally, the air-dried RNA pellet was dissolved in 30-50 μL of RNase-free dH2O. The concentration of the extracted RNA was determined using the Qubit Broad Range assay (Invitrogen, Waltham, USA), while the A280/A260 ratio, measured using the Nanodrop 2000 spectrophotometer (Thermo Scientific, Waltham, USA), served as a quality check for the extracted RNA. Additionally, the quality of the extracted RNA was evaluated by running 500 ng of RNA in a 1% agarose TAE-gel. Quality control, poly-A selection for rRNA removal, library preparation, and multiplexing were performed by Azenta/Genewiz UK according to their standard workflow. Briefly, RNA library preparation was prepared using NEBNext Ultra II Library Prep Kit for Illumina following the manufacturer's protocol (NEB). Firstly, oligo-dt beads were used for mRNA enrichment, and the mRNAs were fragmented for 15 min at 94 oC. First and second strand cDNA were subsequently synthesized. Indexed adapters were ligated to cDNA fragments after adenylation of the 3' end of the cDNA fragments. Limited cycle PCR was used for library amplification. Sequencing libraries were validated using NGS Kit on the Agilent 5300 Fragment Analyzer (Agilent Technologies) and quantified by using Qubit 4.0 Fluorometer (Invitrogen) or equivalent.