RNAseq of submergence response of the flood tolerant Cardamineae tribe
RNAseq of submergence response of the flood tolerant Cardamineae tribe
young leaves including shoot apical meristems were harvested and immediately snap frozen in liquid nitrogen All plant species were grown on a soil mixture until the 10-leaf-stage, about 3-4 weeks after germination, according to established protocols (Müller et al. 2019), under short-day conditions (8 h light, 16 h darkness), 100 µmol photons * m-2 * s-1 and 23 °C. Submergence stress was applied to the plants by immersing them in big, transparent boxes (about 50 l volume) filled with 40 l of tap water 24 h before start of the treatment. Treatment was started 2 hours after start of illumination. Frozen samples were ground for 3 min in 2 ml tubes with zirconia/silica beads using a Mini-Beadbeater (BioSpec Products, Bartlesville, OK, USA) in plates frozen with liquid nitrogen. After grinding, 1 ml lysis binding buffer (Dynabeads mRNA Direct Kit; Thermo Fisher Scientific, Waltham, MA, USA) was added and samples were homogenized with this buffer for an additional 2 min in the Beadbeater. Homogenized samples were centrifuged at 12 000 g for 3 min and loaded onto a homogenizer spin column (Omega Bio-Tek, Norcross, GA, USA) and centrifuged to remove excess tissue. Lysates were transferred to a 96-well plate and stored at −80°C until library preparations. RNA-seq libraries were constructed using breath adapter directional sequencing (BrAD-seq) protocol for non-strand-specific libraries (Townsley et al., 2015). Briefly, 200 μl of lysates was used for messenger RNA (mRNA) isolation, following the manufacturer's instructions (Dynabeads mRNA Direct Kit; Thermo Fisher Scientific). Isolated mRNA was fragmented by reverse transcription (RT) buffer for 90 s at 94°C (RevertAid RT enzyme; Thermo Fisher Scientific), and complementary DNA (cDNA) synthesis was done on the fragmented mRNA. Second-strand synthesis was done according to BrAD-seq protocol and was followed by adapter ligation. PCR enrichment was performed using unique indexed oligos for multiplexing.