title: RNA-seq of Arabidopsis thaliana shoots and roots treated with tunicamycin: a comparison between double mutant (ire1a ire1b) and  wild type genotypes.
design: RNA-seq of Arabidopsis thaliana shoots and roots treated with tunicamycin: a comparison between double mutant (ire1a ire1b) and  wild type genotypes.
library_construction_protocol: Shoot and root samples were seperated using a scalpel. Seedlings were grown in vitro on plates in growth chamber (16h light/8h dark, average light intensity: 1500 lux). Seeds were directly stratified on growth medium (equivalent halh MS + 1% sucrose) for \" days at 4°C, in the dark. Medium was supplemented with either DMSO (0.01% final concentration, control condition) or tunicamycin (final concentration: 100ng/mL, treatment conditions). Total RNA were extracted using the RNeasy Plant Mini kit (#74904, QIAGEN) following manufacturer's instructions. Five µg of total RNA were then treated with Dnase I (RNase-free, EN0525, Fisher, Thermoscientific). DNase I was heat inactivated and treated RNA were precipitated using lithium chloride. Quality of chosen RNA samples to be sequenced was checked using bioanalyzer. RNA samples prepared from shoots displayed RIN values ranging from 6.7 to 7.3 and a ratio RNA 28S/RNA 18S ranging from 1.5 to 1.7. RNA samples prepared from roots displayed RIN values ranging from 7.7 to 10 and a ratio RNA 28S/RNA 18S ranging from 1.7 to 2.1. 1. Take a certain amount of total RNA samples, and use oligo dT beads to enrich mRNA with poly A tail; 2. mRNA molecules were fragmented into small pieces; 3. The fragmented mRNA was synthesized into first strand cDNA using random primers; 4. The second strand cDNA was synthesized with dUTP instead of dTTP; 5. The synthesized cDNA was subjected to end-repair and 3' adenylated. Adaptors were ligated to the ends of these 3' adenylated cDNA fragments; 6. Digest the U-labeled second-strand template with Uracil-DNA-Glycosylase (UDG) and perform PCR amplification; 7. Library quality control; 8. Library circularization; 9. The library was amplified to make DNA nanoball (DNB);