Name: Atha_subm2_II_p
Instrument: Illumina HiSeq 4000
Strategy: ssRNA-seq
Source: TRANSCRIPTOMIC
Selection: RT-PCR
Layout: PAIRED
Construction protocol: 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.